Co-culture of placental stem cells and stem cells from a second source

ABSTRACT

The present invention relates to a combination of placental stem cells and stem or progenitor cells derived from a second source, wherein the combination shows improved engraftment as compared to placental stem cells or stem cells from a second source, alone. The combination is referred to as a combined stem cell population. The invention also provides in vitro and in vivo methods for identifying and producing combined stem cell populations, and models of engraftment. In accordance with the present invention, the placental stem cells may be combined with, e.g., umbilical cord blood-derived stem or progenitor cells, fetal or neonatal stem cells or progenitor cells, adult stem cells or progenitor cells, hematopoietic stem cells or progenitor cells, stem or progenitor cells derived from bone marrow, etc.

This application claims benefit of U.S. Provisional Application No.60/754,692, filed Dec. 29, 2005, the disclosure of which is herebyincorporated by reference herein.

1. INTRODUCTION

The present invention provides in vitro and in vivo methods foroptimizing the ratio of a placenta-derived stem cell population to astem and/or progenitor cell population from a second source to create acombined stem cell population having improved engraftment potential overpopulations of placental stem cells, or stem cells from the secondsource, alone. The present invention also provides combined stem cellpopulations comprising placenta-derived stem cells and stem orprogenitor cells derived from a second source, wherein the combinationshows improved engraftment as compared to placental stem cells or thestem cells from a second source, alone. In accordance with the presentinvention, placenta-derived stem cells may be combined with, e.g.,umbilical cord blood-derived stem or progenitor cells, fetal or neonatalstem cells or progenitor cells, adult stem cells or progenitor cells,hematopoietic stem cells or progenitor cells, stem or progenitor cellsderived from bone marrow, etc. The combined stem cell populations may betransplanted into an individual in need of a transplantation of stemcells, for example, an individual who has undergone myeloablativetherapy and requires re-establishment of an immune and hematopoieticsystem, or an individual having a disease, disorder or conditiontreatable by the introduction to said individual of stem cells. Thecombined stem cell populations may be used to treat any condition thatwould benefit from administration of stem cells, including blooddisorders such as anemia, neurological disorders, immune disorders, andthe like.

2. BACKGROUND OF THE INVENTION

Human stem cells are totipotential, pluripotential or multipotentialprecursor cells capable of generating a variety of mature human celllineages. Stem cells can be employed to repopulate many, if not all,tissues and restore physiologic and anatomic functionality. For example,cell populations containing stem cells have been used in transplants torestore partial or full hematopoietic function in patients who haveundergone ablative therapy.

Recently, Hariri has reported the isolation of stem cells from mammalianplacentas, and the characterization of those stem cells. See Hariri,U.S. Application Publication No. 2002/0123141 “Method of CollectingPlacental Stem Cells,” Hariri, U.S. Application Publication No.2002/0160510 “Renovation and Repopulation of Decellularized Tissues andCadaveric Organs by Stem Cells,” Hariri, U.S. Application PublicationNo. 2003/0032179 “Post-partum Mammalian Placenta, Its Use and PlacentalStem Cells Therefrom,” and Hariri, U.S. Application Publication No.2003/0180269 “Embryonic-like Stem Cells Derived From Post-partumMammalian Placenta, and Uses and Methods of Treatment Using Said Cells”.

Many different types of mammalian stem cells have been characterized.See, e.g., Caplan et al., U.S. Pat. No. 5,486,359 (human mesenchymalstem cells); Hu et al., WO 00/73421 (methods of isolation,cryopreservation, and therapeutic use of human amniotic epithelialcells); Boyse et al., U.S. Pat. No. 5,004,681 (fetal and neonatalhematopoietic stem and progenitor cells); Boyse et al., U.S. Pat. No.5,192,553 (same); Beltrami et al., Cell 114(6):763-766 (2003) (cardiacstem cells); Forbes et al., J. Pathol. 197(4):510-518 (2002) (hepaticstem cells).

The success of transplantation of stem cells is significantly related tothe numbers of engraftable cells administered. The number of engraftablecells in, for example, a unit of cord blood, and the amount of cordblood, that may be obtained from a single donor can vary by two ordersof magnitude. See, e.g., Gluckman, Hematology, American Society ofHematology Education Program Book, 1-14 (1998). Therefore, a need existsfor a method for improvement of the engraftment potential of units ofcord blood, cord blood-derived nucleated cells, or other stem cells,especially prior to transplantation.

3. SUMMARY OF THE INVENTION

The present invention provides a method of determining ratios ofplacenta-derived stem cells to stem cells from a second source toproduce stem cell populations that produce greater numbers ofcolony-forming units, or improved engraftment in vivo, compared toplacental stem cells or stem cells from a second source, alone. Thepresent invention provides methods for enhancing and/or accelerating theengraftment potential of cultures or units of stem cells, progenitorcells, or tissues containing stem or progenitor cells, e.g., cord blood,and combinations thereof. In particular, the invention provides methodsand compositions for enhancing and/or accelerating the engraftmentpotential of a combination of placental stem cells and stem cells from asecond source, e.g., umbilical cord blood or placental blood, or of stemcells derived therefrom. Such populations are referred to herein as“combined stem cell populations”. The invention further provides in vivouses for the combined stem cell populations. In a preferred embodiment,the placental stem cells are placental stem cells contained within apopulation of cells obtained from placental perfusate.

In one embodiment, the invention provides a method of identifying aratio of placental stem cells to stem cells from a second source,comprising identifying a ratio of placental stem cells to stem cellsfrom a second source in a total number of cells that, when saidplacental stem cells and stem cells from a second source are culturedtogether for a time and under conditions sufficient to allow theformation of colony-forming units, produces a greater number ofcolony-forming units than a number of placental stem cells or a numberof stem cells from a second source, equivalent to said total number ofcells, alone, thereby identifying said combination as a combined stemcell population. In a specific embodiment, said combined stem cellpopulation improves engraftment in an individual in need of stem cellswhen said combined stem cell population is transplanted into saidindividual, compared to the transplantation of a number of placentalstem cells equivalent to said number of cells, or stem cells from asecond source equivalent to said number of cells, alone.

In another embodiment, the invention provides a method of identifying acombined stem cell population comprising contacting in vitro placentalstem cells with stem cells from a second source in a plurality ofratios, for a time and under conditions that allow the formation ofcolony-forming units, and identifying a ratio within said plurality ofratios that produces the greatest number of colony-forming units,wherein said placental stem cells and said stem cells from a secondsource, when combined in said ratio, are identified as a combined stemcell population. In a specific embodiment, said combined stem cellpopulation improves engraftment in an individual in need of stem cellswhen said combined stem cell population is transplanted into saidindividual.

In more specific embodiments, said combined stem cell populationimproves engraftment in an individual in need of stem cells at least, orat, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or 21 days post-transplant. In another more specific embodiment, saidcombined stem cell population improves engraftment in an individual inneed of stem cells at least, or at, more than 21 days post-transplant.In specific embodiments, said combined stem cell population improvesengraftment in an individual in need of stem cells at least, or at, morethan 25, 30, 35, 40, 45, 50, 55 weeks, or 1 year or longerpost-transplant.

In another specific embodiment, said contacting comprises culturing saidplacental stem cells and said stem cells from a second source in thesame physical space. In another specific embodiment, said contactingcomprises culturing said placental stem cells and said stem cells from asecond source in separate physical spaces in shared culture medium.

In another embodiment, said stem cells from a second source are stemcells derived from cord blood. In another embodiment, placental stemcells comprise CD34⁺ cells, for example, CD34⁺CD38⁺ cells and/orCD34⁺CD38⁻ cells. In another embodiment, placental stem cells comprisecells that express one or more of markers CD10, CD29, CD44, CD54, CD90,CD73 or CD105, and lack one or more of markers CD34, CD38, CD45, SSEA3and SSEA4. In another embodiment, placental stem cells comprise cellsthat are positive for CD10, CD29, CD44, CD54, CD90, CD73 or CD105, andnegative for CD34, CD38, CD45, SSEA3 and SSEA4. In another embodiment,placental stem cells comprise cells that comprise one or more of markersCD10, CD29, CD44, CD54, CD90, CD73 and CD105, and lack one or more ofmarkers CD34, CD38, CD45, SSEA3 and SSEA4. In another embodiment,placental stem cells comprise cells that are positive for CD10, CD29,CD44, CD54, CD90, CD73 and CD105, and negative for CD34, CD38, CD45,SSEA3 and SSEA4. In another embodiment, said placental stem cellscomprise CD34⁻ cells. In a specific embodiment, said placental stemcells are CD34⁻CD38⁻ placental stem cells. In another embodiment, saidplacental stem cells are OCT-4⁺ or ABC-p⁺. In a more specificembodiment, said placental stem cells are OCT-4⁺ and ABC-p⁺. In anotherembodiment, said placental stem cells comprise cells that are positivefor CD10, CD29, CD33, CD44, CD73, CD105, CD117, and CD133, and negativefor CD34 or CD45. In a more specific embodiment, said placental stemcells comprise cells that are HLA-ABC⁺. In a more specific embodiment,said placental stem cells comprise cells that are HLA-ABC⁻. In a morespecific embodiment, said placental stem cells comprise cells that areHLA-DR⁺. In a more specific embodiment, said placental stem cellscomprise cells that are HLA-DR⁻. In another specific embodiment, theplacental stem cells comprise cells that are CD200⁺ and HLA-G⁺. Inanother specific embodiment, the placental stem cells comprise cellsthat are CD73⁺, CD105⁺ and CD200⁺. In another specific embodiment, theplacental stem cells comprise cells that are CD200⁺ and OCT-4⁺. Inanother specific embodiment, the placental stem cells comprise cellsthat are CD73⁺, CD105⁺ and facilitate the formation of embryoid-likebodies in a population of isolated placental cells comprising said stemcells, when said population is cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, theplacental stem cells comprise cells that are CD73⁺, CD105⁺ and HLA-G⁺.In another specific embodiment, the placental stem cells comprise cellsthat are OCT-4⁺ and facilitate the formation of embryoid-like bodies ina population of isolated placental cells comprising said stem cells,when said population is cultured under conditions that allow theformation of embryoid-like bodies.

In a specific embodiment, said placental stem cells are obtained from asingle placenta. In another specific embodiment, said placental stemcells are obtained from a plurality of placentas. In another specificembodiment, said placental stem cells are obtained from placentalperfusate. In another specific embodiment, said placental stem cells areobtained from said placenta by perfusion of said placenta with aperfusion solution. In a more specific embodiment, said perfusionsolution comprises a protease or a mucolytic enzyme. In another specificembodiment, said placental stem cells are obtained by physicaldisruption of the placenta, or a part of the placenta. In a morespecific embodiment, said physical disruption comprises contacting saidplacenta with a protease or mucolytic enzyme. In an even more specificembodiment, said protease is a collagenase (e.g., collagenase I,collagenase IV), trypsin (e.g., trypsin-EDTA), elastase, dispase, or acombination thereof. In another even more specific embodiment, saidmucolytic enzyme is hyaluronidase.

In another specific embodiment, said stem cells from a second source arecord blood-derived stem cells. In a more specific embodiment, said cordblood-derived cells are hematopoietic stem cells. In another morespecific embodiment, said cord blood-derived cells are non-hematopoieticstem cells. In another specific embodiment, said placental stem cellsand stem cells from a second source are combined in suspension. Inanother specific embodiment, the method additionally comprises adding tosaid combination a bioactive molecule. In a more specific embodiment,said bioactive molecule is a cytokine or growth factor.

The present invention also provides a combined stem cell populationcomprising a number of cells in vitro, said number of cells comprisingplacental stem cells and stem cells from a second source, wherein saidcombined stem cell population, when cultured for a time and underconditions that allow the formation of colony-forming units, producesmore colony-forming units than a number of placental stem cellsequivalent to the number of cells in the combined stem cell populationor a number of stem cells from a second source equivalent to the numberof cells in the combined stem cell population, alone. The presentinvention further provides a combined stem cell population comprising anumber of placental stem cells and stem cells from a second source invitro, wherein transplantation of said combined stem cell populationenhances engraftment of said stem cells compared to transplantation of anumber of said placental stem cells equivalent to the number of cells inthe combined stem cell population or a number of stem cells from asecond source equivalent to the number of cells in the combined stemcell population, alone. In another specific embodiment, the combinedstem cell population comprises said placental stem cells and said stemcells from a second source in a ratio, out a plurality of ratios, that,when cultured under conditions allowing the formation of colony formingunits, produces the most colony forming units. In a specific embodiment,said stem cells from a second source are cord blood stem cells, bonemarrow stem cells, hematopoietic stem cells, or mesenchymal stem cells.In a more specific embodiment, said hematopoietic stem cells are cordblood hematopoietic stem cells. In another more specific embodiment,said hematopoietic stem cells are CD34⁺ cells. In another specificembodiment, said placental stem cells comprise CD34⁺ cells. In anotherspecific embodiment, said placental stem cells comprise CD34⁻ cells. Inanother specific embodiment, said placental stem cells comprise cellsthat are OCT4⁺ or ABC-p⁺. In another specific embodiment, said placentalstem cells comprise cells that are CD34⁺ and cells that are OCT4⁺ orABC-p⁺. In another specific embodiment, said placental stem cells arecontained within placental perfusate substantially lacking red bloodcells and cellular debris. In another specific embodiment, the placentalstem cells comprise, or are, placental stem cells isolated fromplacental perfusate. In another specific embodiment, the placental stemcells are contained within total nucleated cells from placentalperfusate. In another specific embodiment, said placental stem cells arecontained within a population of cells obtained from placentalperfusate. In another specific embodiment, said composition comprisesplacental cells isolated from enzyme-digested placental tissue. Inanother specific embodiment, said placental stem cells and said stemcells from a second source are obtained from the same individual. Inanother specific embodiment, said placental stem cells and said stemcells from a second source are obtained from different individuals. Inanother specific embodiment, said placental stem cells are derived froma plurality of placentas. In another specific embodiment, said stemcells from a second source are obtained from a plurality of individuals.

In another embodiment, placental stem cells in said combined stem cellpopulation comprise CD34⁺ cells, for example, CD34⁺CD38⁺ cells and/orCD34⁺CD38⁻ cells. In another embodiment, placental stem cells comprisecells that express one or more of markers CD10, CD29, CD44, CD54, CD90,CD73 or CD105, and lack one or more of markers CD34, CD38, CD45, SSEA3and SSEA4. In another embodiment, placental stem cells comprise cellsthat are positive for CD10, CD29, CD44, CD54, CD90, CD73 or CD105, andnegative for CD34, CD38, CD45, SSEA3 and SSEA4. In another embodiment,placental stem cells comprise cells that comprise one or more of markersCD10, CD29, CD44, CD54, CD90, CD73 and CD105, and lack one or more ofmarkers CD34, CD38, CD45, SSEA3 and SSEA4. In another embodiment,placental stem cells comprise cells that are positive for CD10, CD29,CD44, CD54, CD90, CD73 and CD105, and negative for CD34, CD38, CD45,SSEA3 and SSEA4. In another embodiment, said placental stem cellscomprise CD34⁻ cells. In a specific embodiment, said placental stemcells are CD34⁻CD38⁻ placental stem cells. In another embodiment, saidplacental stem cells are OCT-4⁺ or ABC-p⁺. In a more specificembodiment, said placental stem cells are OCT-4⁺ and ABC-p⁺. In anotherembodiment, said placental stem cells comprise cells that are positivefor CD10, CD29, CD33, CD44, CD73, CD105, CD117, and CD133, and negativefor CD34 or CD45. In a more specific embodiment, said placental stemcells comprise cells that are HLA-ABC⁺. In a more specific embodiment,said placental stem cells comprise cells that are HLA-ABC⁻. In a morespecific embodiment, said placental stem cells comprise cells that areHLA-DR⁺. In a more specific embodiment, said placental stem cellscomprise cells that are HLA-DR⁻. In another specific embodiment, theplacental stem cells comprise cells that are CD200⁺ and HLA-G⁺. Inanother specific embodiment, the placental stem cells comprise cellsthat are CD73⁺, CD105⁺ and CD200⁺. In another specific embodiment, theplacental stem cells comprise cells that are CD200⁺ and OCT-4⁺. Inanother specific embodiment, the placental stem cells comprise cellsthat are CD73⁺, CD105⁺ and facilitate the formation of embryoid-likebodies in a population of isolated placental cells comprising said stemcells, when said population is cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, theplacental stem cells comprise cells that are CD73⁺, CD105⁺ and HLA-G⁺.In another specific embodiment, the placental stem cells comprise cellsthat are OCT-4⁺ and facilitate the formation of embryoid-like bodies ina population of isolated placental cells comprising said stem cells,when said population is cultured under conditions that allow theformation of embryoid-like bodies.

In another embodiment, placental stem cells, or stem cells from a secondsource, in said combined stem cell population comprise CD34⁺ cells thatare positive for aldehyde dehydrogenase (ALDH). Such cells demonstratedetectable levels of ALDH activity in an ALDH assay. Thus, in variousembodiments, a combined stem cell population of the invention comprisesCD34+ stem cells, where at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95%of the CD34⁺ stem cells are ALDH⁺.

The present invention also provides pharmaceutical compositions thatcomprise combined stem cell populations, e.g., placental perfusate,placental enzymatic digestate, or placental stem cells derivedtherefrom, combined with umbilical cord blood or umbilical cordblood-derived stem cells, in a pharmaceutically-acceptable carrier. Invarious specific embodiments, the placental stem cells in said combinedstem cell population can be derived from a single donor, or from aplurality of donors; the stem cells from a second source may be derivedfrom a single donor, or from a plurality of donors; or both theplacental stem cells and the stem cells from a second source may bederived from single donor, or from a plurality of donors. The combinedstem cell populations useful in the methods of the invention maycomprise stem cell populations that are partially or completely non-HLAmatched to an intended recipient, as well as stem or progenitor cellpopulations that are completely HLA-matched to an intended recipient.

Combined stem cell populations, e.g., umbilical cord blood supplementedwith placental perfusate or placental perfusate-derived stem and/orprogenitor cells in an optimum ratio, have a multitude of uses,including prophylactic, therapeutic and diagnostic uses. In oneembodiment of the invention, the combined stem cell populationscomprising placental stem cells and stem cells from a second source areused to renovate and repopulate tissues and organs, thereby replacing orrepairing diseased tissues, organs or portions thereof. In anotherembodiment, the combination stem cell populations comprising placentalstem cells and stem cells from a second source are used to promotere-establishment of hematopoiesis in individuals that have undergonepartial or complete myeloablation. In another embodiment, thecombination stem cell populations are used to promote re-establishmentof hematopoiesis in an individual that has been exposed to a lethal orsub-lethal dose of radiation.

The present invention also provides methods of transplantation, and oftreating an individual in need thereof, by administration of a combinedstem cell population, comprising transplanting to said individual anumber of placental stem cells and stem cells from a second source in aratio, wherein said combined stem cell population exhibits improvedengraftment as compared to transplanting a number of placental stemcells equivalent to the number of cells in the combined stem cellpopulation or a number of stem cells from a second source equivalent tothe number of cells in the combined stem cell population, alone. In morespecific embodiments, transplantation of said combined stem cellpopulation improves engraftment in an individual in need of stem cellsat least, or at, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or 21 days post-transplant, compared to transplantationof a number of placental stem cells equivalent to the number of cells inthe combined stem cell population or stem cells from a second sourceequivalent to the number of cells in the combined stem cell population,alone. In another more specific embodiment, said combined stem cellpopulation improves engraftment in an individual in need of stem cellsmore than 21 days post-transplant.

In a more specific embodiment, said ratio is a ratio in a total numberof cells that produces in vitro more colony-forming units than either anumber of placental stem cells or stem cells from a second source,equivalent to said total number of cells, alone, under conditions thatallow the formation of colony-forming units. In another more specificembodiment, said ratio is the ratio in a plurality of ratios ofplacental stem cells and stem cells from a second source that, whencombined in vitro under conditions that allow the formation ofcolony-forming units, produces the greatest number of colony-formingunits. That is, if X is the number of placental stem cells plus the stemcells from a second source, in such an embodiment, the ratio ofplacental stem cells to stem cells from a second source produces invitro more colony-forming units than either X placental stem cellsalone, or X stem cells from a second source, alone.

The invention further provides for the assembly of a bank ofHLA-characterized placenta-derived stem cells for use in producingcombined stem cell populations of the invention. In one embodiment, theinvention provides a stem cell bank comprising a plurality of units ofplacenta-derived stem cells, wherein said placenta-derived stem cellsare identified by at least one HLA marker. In a specific embodiment,said placenta-derived stem cells are isolated from placental perfusate.In another specific embodiment, said placenta-derived stem cells arecontained within a population of nucleated cells isolated from placentalperfusate. In another specific embodiment, said placenta-derived stemcells are CD34⁺ stem cells. In another specific embodiment, saidplacenta-derived stem cells are positive for CD73 or CD105, or are boundby antibodies SH2, SH3 or SH4. In another specific embodiment, said stemcell bank additionally comprises a plurality of units of placental bloodor umbilical cord blood. In another specific embodiment, at least oneunit of said plurality of units of placental blood or umbilical cordblood is identified by an HLA marker shared by one of said plurality ofunits of placenta-derived stem cells. In another specific embodiment, amajority of units within said plurality of units of placental blood orumbilical cord blood is identified by an HLA marker shared by a majorityof units within said plurality of units of placenta-derived stem cells.

3.1 Definitions

As used herein, the term “exsanguinated” or “exsanguination,” when usedwith respect to the placenta, refers to the removal and/or draining ofsubstantially all cord blood from the placenta.

As used herein, “passage,” with respect to cell culture, means thealiquoting of a plurality of cells from one culture into a separatecontainer to start a new culture of cells. Typically, passagingcomprises the aliquoting of, e.g., 10⁴-10⁵ cells from one culture in onecontainer into fresh medium in a separate container. Cells are typicallypassaged when a culture of cells approaches confluency, that is, when amonolayer of adherent cells forms a single layer over the entire areaavailable for growth.

As used herein, the term “perfuse” or “perfusion” refers to the act ofpassing a fluid through the vasculature of a placenta with a forcesufficient to collect a plurality of placental cells. As used herein,the term “placental perfusate” refers to the fluid collected followingits passage through a placenta, including cells that have been collectedfrom the placenta during perfusion.

As used herein, the terms “placental blood” and “umbilical cord blood”are equivalent.

As used herein, the terms “placental stem cell” and “placenta-derivedstem cell” are equivalent.

As used herein, the term “placental stem cell” refers to a stem cellthat is obtained from or derived from a mammalian placenta, or a portionthereof (e.g., amnion, chorion, and the like) regardless of morphology,cell surface markers, etc., but does not encompass a trophoblast. Thephrase encompasses a stem cell obtained directly from a placenta, e.g.,as part of a population of placental cells in placental perfusate ordigested placental tissue (digestate), or a stem cell that is part of apopulation of placental cells that has been expanded and/or passaged oneor more times. The term does not, however, encompass stem cells derivedsolely from another tissue, e.g., placental blood or umbilical cordblood. The placenta comprises stem cell populations having, anddistinguishable from each other by, for example, distinct sets ofmarkers.

As used herein, the term “positive,” in reference to a stem cell marker,means that the marker is present in a detectably higher amount, ordetectably higher level, than the amount or level of said marker in areference non-stem cell, e.g., a fibroblast. More generally, a cell is“positive” for a marker when the cell can be differentiated from one ormore other cell types on the basis of the presence of that marker in oron the cell.

As used herein, “stem cell from a second source” means any mammalianstem cell (including progenitor cells) from a source other than amammalian placenta.

As used herein, the term “stem cell” encompasses stem cells andprogenitor cells.

As used herein, the term “unit,” when applied to cord blood or placentalblood, indicates a single collection of blood from a single donor, orthe nucleated cells, or the stem cells, obtainable from such acollection. Typically, the volume of blood from a single donor rangesfrom about 50 to about 150 ml of blood. The term “unit,” when applied toplacental perfusate, means the volume of perfusion fluid used to collectplacental stem and progenitor cells from a single placenta, or thenucleated cells, or the stem cells, obtainable from such a volume ofperfusion solution. The volume of placental perfusate in a unit istypically from about 100-500 ml to about 1000 ml.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D: Summary of FACS analysis of engrafted human cells in micebone marrow using CD45 antibodies in two independent experiments. (A):First experiment, CD45+ cells present in bone marrow at 3 weeks forumbilical cord blood cells only (UCB), placental perfusate cells only(PP) or umbilical cord cells combined with placental perfusate cells(UCB+PP). X-axis: numbers of cells per transplantation. (B): Firstexperiment, CD45⁺ cells at 10 weeks post-transfusion. (C): Secondexperiment, CD45⁺ cells in bone marrow at 3 weeks post-transfusion. (D):Second experiment, CD45⁺ cells in bone marrow at 10 weekspost-transfusion.

FIG. 2: FACS analysis of engrafted human cells expressing lymphomyeloidcell markers in NOD/SCID mice. Co-expression of CD45⁺ with CD19 (leftbar in each category); CD33 (middle bar); or CD7 (right bar). X-axis:numbers of cells per transplantation. UCB=transplantation of umbilicalcord blood cells only; PP=transplantation of placental perfusate cellsonly. UCB+PP=transplantation of umbilical cord cells combined withplacental perfusate cells.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides combinations of (1) placental stem cells,e.g., placental stem cells in human placental perfusate, placental stemcells in placental enzymatic digestate, isolated placental stem and/orprogenitor cells, and the like; and (2) stem cells from a second source,in a total number of cells, wherein the placental stem cells and stemcells from the second source are present in the combination in a ratiothat produces a greater number of colony-forming units compared to anumber of colony-forming units produced by placental stem cells or bystem cells from a second source, equivalent to said total number ofcells, alone. The invention further provides combinations of placentalstem cells and stem cells from a second source that enhance engraftmentin vivo compared to the number of colony-forming units produced by anumber of placental stem cells equivalent to the number of cells in saidcombination, or a number of stem cells from a second source equivalentto the number of cells in said combination, alone. The present inventionfurther provides methods of identifying such ratios, and suchcombinations, and methods of using the combined stem cell populations.

5.1 Optimizing Combinations of Placental Stem Cells and Stem Cells froma Second Source

5.1.1 In Vitro Assay

The invention provides in vitro co-culture methods for identifying acombination of placental stem cells and stem cells from a second sourcethat has improved engraftment potential as compared to a number ofeither placental stem cells or stem or progenitor cells from a secondsource, equivalent to the number of cells in said combination, alone.The in vitro co-culture assay thus identifies ratios of placental stemcells to stem cells from a second source that improve the number ofcolony-forming units, and engraftment, in a non-cell number-dependentmanner.

In one embodiment, for example, the invention provides a method ofidentifying a ratio of placental stem cells to stem cells from a secondsource, comprising identifying a ratio of placental stem cells to stemcells from a second source in a total number of cells that, when saidplacental stem cells and stem cells from a second source are culturedtogether for a time and under conditions that allow the formation ofcolony-forming units, produces a greater number of colony-forming unitsthan a number of placental stem cells or stem cells from a secondsource, equivalent to the number of cells in said total number of cells,alone. In another embodiment, where several ratios are compared, theinvention provides a method of identifying a ratio of placental stemcells and stem cells or progenitor cells from a second source in a totalnumber of cells, comprising contacting a population of said placentalstem cells in vitro with a population of said stem cells from a secondsource in a plurality of ratios for a time and under conditionssufficient to allow the formation of colony-forming units, andidentifying a ratio within said plurality of ratios that yields thegreatest number of colony-forming units. In a specific embodiment, saidratio improves engraftment into a recipient as compared to engraftmentby a number of placental stem cells or stem cells from a second source,equivalent to the number of cells in said total number of cells, alone.In more specific embodiments, said combined stem cell populationimproves engraftment in an individual in need of stem cells for at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or21 days post-transplant. In another more specific embodiment, saidcombined stem cell population improves engraftment in an individual inneed of stem cells at a time more than 21 days post-transplant.

5.1.1.1 Placenta-Derived Stem Cells

Placenta-derived stem cells useful in the methods and compositions ofthe invention include, for example, embryonic-like cells, pluripotentcells, multipotent cells, committed progenitor cells, hematopoieticprogenitor cells, and mesenchymal-like stem cells from placenta. In oneembodiment, the placenta-derive stem cells are contained within, or arederived from, placental perfusate.

Placenta-derived stem cells used in the methods of the invention can bederived from a single placenta, or from a plurality of placentas, andmay be obtained by any method. Placenta-derived stem cells can beobtained by, for example, perfusion, as disclosed in U.S. ApplicationPublication Nos. 2002/0123141 and 2003/0032179, the disclosures of eachof which are incorporated herein by reference. Such perfusion can beperfusion by the pan method, wherein perfusion liquid is forced throughthe placental vasculature and perfusion fluid that exudes from theplacenta, typically the maternal side, is collected in a pan containingthe placenta. Perfusion can also be a closed-circuit perfusion, whereinperfusion fluid is passed through, and collected from, only the fetalvasculature of the placenta. In a specific embodiment, such perfusioncan be continuous, that is, perfusion fluid that has been passed throughthe placenta, and which comprises a plurality of placental cells, ispassed through a second time, or a plurality of times, prior toisolation of placental cells.

Placenta-derived stem cells may also be obtained by physical orenzymatic disruption of the placenta using, e.g., proteases and/or othertissue-disruptive enzymes to disrupt the multicellular structure of theplacenta. Such proteases may include neutral proteases ormetalloproteases, e.g., collagenase, dispase, trypsin, elastase, and thelike. Placental stem cells may also be obtained by physical disruptionof the placenta using, e.g., mucolytic enzymes, for example,hyaluronidase.

The isolated perfused placenta of the invention provides a source oflarge quantities of stem cells enriched for CD34⁺ stem cells, e.g.,CD34⁺CD38⁻ stem cells, e.g., CD34⁺, CD38⁻, lin⁻ stem cells, and CD34⁻stem cells, e.g., CD34⁻CD38⁺ stem cells. The first collection of bloodfrom the placenta is referred to as cord blood which containspredominantly CD34⁺CD38⁺ hematopoietic progenitor cells. Within thefirst twenty-four hours of post-partum perfusion, high numbers (e.g.,1×10⁵ to about 2×10⁷) of CD34⁺CD38⁻ hematopoietic progenitor cells maybe isolated from the placenta, along with high concentrations ofCD34⁻CD38⁺ cells. After about twenty-four hours of perfusion, highnumbers (e.g., 1-10 million) of CD34⁻CD38⁻ cells can be isolated fromthe placenta along with the aforementioned cells. An isolated placentathat has been perfused for twenty-four hours or more provides a sourceof large quantities of stem cells enriched for CD34⁻CD38⁻ stem cells.

In another embodiment, the combined stem cell populations of theinvention comprise CD34⁺ placental stem cells that are positive foraldehyde dehydrogenase (ALDH). Such cells demonstrate detectable levelsof ALDH activity in an ALDH assay. Such assays are known in the art(see, e.g., Bostian and Betts, Biochem. J., 173, 787, (1978)). In aspecific embodiment, said ALDH assay uses ALDEFLUOR® (Aldagen, Inc.,Ashland, Oreg.) as a marker of aldehyde dehydrogenase activity. Thus, invarious embodiments, a combined stem cell population of the inventioncomprises CD34+ stem cells, where at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or atleast 95% of the CD34⁺ stem cells are ALDH⁺.

At least one class of human placental stem cells has characteristics ofembryonic stem or germ cells. For example, stem cells of this class areSSEA3⁻ (stage-specific embryonic antigen 3), SSEA4⁻, OCT-4⁺ (a stem celltranscription factor) and ABC-p⁺ (ATP-binding cassette (ABC) transporterprotein), a marker profile exhibited by pluripotent stem cells that havenot yet undergone differentiation. Thus, the methods and compositions ofthe invention can use or comprise non-embryonic, placental stem cellsthat are, e.g., SSEA3⁻, SSEA4⁻, OCT-4⁺ or ABC-p⁺. Preferably, theplacental stem cells are OCT-4⁺ABC-p⁺, and, even more preferably, areSSEA3⁻SSEA4⁻OCT-4⁺ABC-p⁺. In another embodiment, the inventionencompasses the use of placental stem cells positive for at least one ofCD10, CD29, CD44, CD54, CD90, CD73 or CD105, or negative for at leastone of CD34, CD38, or CD45. In another embodiment, the methods andcompositions of the invention can use or comprise placental stem cellshaving or positive for CD10, CD29, CD44, CD54, CD90, CD73 or CD105, andlacking or negative for CD34, CD38, or CD45. In another embodiment, themethods and compositions of the invention can use or comprise placentalstem cells positive for at least one of CD10, CD29, CD44, CD54, CD90,CD73 or CD105, or negative for at least one of CD34, CD38, or CD45. Inanother embodiment, the invention encompasses the use of placental stemcells having or positive for CD10, CD29, CD44, CD54, CD90, CD73 orCD105, and lacking or negative for CD34, CD38, or CD45.

In one embodiment, placental stem cells used in the methods andcompositions of the invention are identified by the presence of themarkers CD10, CD29, CD44, CD54, CD90, CD105 (SH2), CD73 (SH3, SH4),OCT-4, and/or ABC-p, and/or the absence of the markers CD34, CD38, CD45,SSEA3, or SSEA4. In a specific embodiment, the placental stem cells areCD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45−, CD54⁺, CD73⁺, CD90⁺, CD105⁺,SH2⁺, SH3⁺, SH4⁺, SSEA3⁻, SSEA4⁻, OCT-4⁺, and ABC-p⁺. In anotherspecific embodiment, the placental stem cells are CD200⁺ and HLA-G⁺. Inthis context, “SH2⁺”, “SH3⁺” and “SH4⁺” mean that a stem cell is boundby antibody SH2, SH3, or SH4, respectively. In another specificembodiment, the placental stem cells are CD73⁺, CD105⁺ and CD200⁺. Inanother specific embodiment, the placental stem cells are CD200⁺ andOCT-4⁺. In another specific embodiment, the placental stem cells areCD73⁺, CD105⁺ and facilitate the formation of embryoid-like bodies in apopulation of isolated placental cells comprising said stem cells, whensaid population is cultured under conditions that allow the formation ofembryoid-like bodies. In another specific embodiment, the placental stemcells are CD73⁺, CD105⁺ and HLA-G⁺. In another specific embodiment, theplacental stem cells are OCT-4⁺ and facilitate the formation ofembryoid-like bodies in a population of isolated placental cellscomprising said stem cells, when said population is cultured underconditions that allow the formation of embryoid-like bodies. As usedherein, “embryoid-like bodies” refers to three-dimensional clusters ofdifferentiating, and differentiated, cells that emerge from the adherentstem cell layer.

In another embodiment, the human placental stem cells do not express MHCClass 2 antigens.

Populations of placental perfusate-derived stem cells, in oneembodiment, comprise trophoblasts.

Cell markers, e.g., stem cell markers and cell surface markers, can beroutinely determined according to methods well known in the art, e.g. byflow cytometry or fluorescence-activated cell sorting (FACS) analysis bywashing and staining with an anti-cell surface marker antibody labeledwith an appropriate fluorophore. For example, to determine the presenceof CD34 or CD38, cells may be washed in PBS and then double-stained withanti-CD34 phycoerythrin and anti-CD38 fluorescein isothiocyanate (BectonDickinson, Mountain View, Calif.). The cells would then be analyzedusing a standard flow cytometer. Alternatively, intra-cellular markerscan also be examined via standard methodology. Antibody/fluorophorecombinations to specific markers include, but are not limited to,fluorescein isothiocyanate (FITC) conjugated monoclonal antibodiesagainst HLA-G (available from Serotec, Raleigh, N.C.), CD10 (availablefrom BD Immunocytometry Systems, San Jose, Calif.), CD44 (available fromBD Biosciences Pharmingen, San Jose, Calif.), and CD105 (available fromR&D Systems Inc., Minneapolis, Minn.); phycoerythrin (PE) conjugatedmonoclonal antibodies against CD44, CD200, CD117, and CD13 (BDBiosciences Pharmingen); phycoerythrin-Cy7 (PE Cy7) conjugatedmonoclonal antibodies against CD33 and CD10 (BD Biosciences Pharmingen);allophycocyanin (APC) conjugated streptavidin and monoclonal antibodiesagainst CD38 (BD Biosciences Pharmingen); and Biotinylated CD90 (BDBiosciences Pharmingen). Other antibody/label combinations that can beused include, but are not limited to, CD133-APC (Miltenyi), KDR-Biotin(CD309, Abcam), CytokeratinK-Fitc (Sigma or Dako), HLA ABC-Fitc (BD),HLA DRDQDP-PE (BD), β-2-microglobulin-PE (BD), CD80-PE (BD) and CD86-APC(BD), CD45-PerCP (peridin chlorophyll protein); CD44-PE; CD19-PE; CD10-F(fluorescein); HLA-G-F and 7-amino-actinomycin-D (7-AAD); HLA-ABC-F; andthe like.

Placental stem cells, e.g., placental stem cells contained in placentalperfusate, can be used immediately after collection, or can be culturedfor a period of time prior to assaying or administration to anindividual in a combined stem cell population. For example, in oneembodiment, the stem cells can be cultured in medium comprising Notchagonist, e.g., a deletion form of a Notch protein consisting essentiallyof the intracellular domain of the Notch protein, or a Delta protein.See U.S. 2004/0067583.

5.1.1.2 Stem Cells from a Second Source

The methods and compositions described herein use placental stem cellsin combination with stem cells from a second source, that is, stem cellsfrom any source other than a mammalian placenta. Stem cells from asecond source can comprise one or more types of stem cells, such asembryonic stem cells, embryonic germ cells, adult stem cells,mesenchymal stem cells, hematopoietic stem cells, non-hematopoietic stemcells, bone marrow-derived stem cells, neural stem cells, cardiac stemcells, ocular stem cells, epithelial stem cells, endothelial stem cells,hepatic stem cells, pulmonary stem cells, muscle stem cells, intestinalstem cells, and the like. Stem cells from a second source can be stemcells isolated from the second, non-placental source, or can be tissuecomprising the stem cells. As for the placenta, stem cells can beisolated by perfusion of the organ(s) comprising the stem cells, or bytissue disruption and/or enzymatic digestion of the organ(s) comprisingthe stem cells. Stem cells from a second source can be, e.g., stem cellsderived solely from umbilical cord, or solely from amniotic fluid.

Stem cells from a second source may be obtained by providing a sample ofa relevant tissue, and isolating stem cells from the tissue using one ormore cell surface markers. For example, hematopoietic stem cells may beobtained from blood (e.g., peripheral blood, placental blood, umbilicalcord blood) or from bone marrow by obtaining a sample of blood or bonemarrow, isolating mononuclear cells from the blood or bone marrow, andseparating CD34⁺ cells from the isolated mononuclear cells. Suchseparation may be accomplished by methods routine in the art, e.g. usingapherisis, followed by separation using magnetic beads or a columncomprising one or more antibodies to the cell surface marker, e.g., CD34or CD200; fluorescence-activated cell sorting (FACS), and the like. Forblood, the stem cells can be provided in a population of total nucleatedcells (TNC) from the blood, e.g., total nucleated cells from peripheralblood, placental blood, umbilical cord blood, and the like.

Stem cells from other tissues may be isolated in a similar manner.Mesenchymal stem cells may be isolated from, e.g., bone marrow byisolation of cells positive for CD73, CD105 and/or CD45 (see, e.g., U.S.Pat. No. 6,387,367). Ocular (limbal) stem cells may be obtained from thecornea by obtaining corneal cells and isolating SSEA-4⁺ cells (see,e.g., U.S. Application Publication No. 2005/0186672). Hepatic stem cellsmay be obtained from liver, particularly fetal liver, samples, byselecting cells expressing CD14, CD34, CD38, ICAM, CD45, CD117,glycophorin A, connexin 32, osteopontin, bone sialoprotein, collagen I,collagen II, collagen III, collagen IV, or combinations thereof (see,e.g., U.S. Application Publication No. 2005/0148072). Muscle stem cellsmay be obtained from muscle tissue by selecting CD34⁺CD45⁻ cells that donot express other hematopoietic cell markers (see, e.g., U.S.Application Publication No. 2005/0079606). Cardiac stem cells may beisolated from cardiac tissue by selecting c-kit⁻CD31⁺CD38⁺ cells (see,e.g., U.S. Application Publication No. 2004/0126879). Isolation of stemcells may be accomplished using other known characteristics or markers,as well.

In one embodiment, said stem cells from a second source are cord bloodstem cells. In specific embodiments, the cord blood stem cells are CD34⁺stem cells, e.g., CD34⁺, CD38⁺ stem cells, CD34⁺, CD38⁻ stem cells,CD34⁺, CD38⁻, lin⁻ stem cells, and the like. In a specific embodiment,the CD34⁺ stem cells from a second source are ALDH⁺. Cord blood itself,or stem and/or progenitor cells obtained from cord blood, can be used inthe methods of the invention. In a specific embodiment, said cordblood-derived cells comprise hematopoietic stem cells, where thecombined stem cell population is to be used for hematopoieticengraftment. The stem cells from a second source may be derived from asingle donor, or from a plurality of donors in equal or unequal amounts.Stem cells from a plurality of second (that is, non-placental) sourcesmay be combined with placental stem cells, and used for the methods andcompositions of the present invention.

Stem cells from a second source, e.g., hematopoietic stem cells from asecond source, can be used immediately after collection, or can becultured for a period of time prior to assaying or administration to anindividual in a combined stem cell population. For example, in oneembodiment, the stem cells can be cultured in medium comprising Notchagonist, e.g., a deletion form of a Notch protein consisting essentiallyof the intracellular domain of the Notch protein, or a Delta protein.See U.S. 2004/0067583

5.1.1.3 Assay Parameters

Once a population of placental stem cells and a population of stem cellsfrom a second source are obtained, the cells can be combined in an invitro co-culture, or colony-forming, assay to determine if the number ofstem cells in a particular combination produces more colony-formingunits than a number of placental stem cells or stem cells from a secondsource, equivalent to the number of cells in said combination, alone.Any such combination of placental stem cells and stem cells from asecond source in a ratio that produces more colony forming units thaneither placental stem cells or stem cells from a second source alone,for equivalent numbers of cells, is identified as a combined stem cellpopulation of the invention.

The identification of a combined stem cell population can use any colonyforming unit assay commonly used and known in the art, provided theassay allows for the proliferation and differentiation of stem cellsfrom placenta and from a second source, for example, colony formingassays provided by StemCell Technologies, Inc. Such an assay may use,e.g., MESENCULT™ medium (Stem Cell Technologies, Inc., Vancouver BritishColumbia). The identification of combined stem cell populations can usecells that are freshly-prepared, or thawed from frozen stocks, or both.Preferably, both the placental stem cells and stem cells from a secondsource are in suspension when combined for co-culture. Placental stemcells, and stem cells from a second source, may be assessed forviability, proliferation potential, and longevity using standardtechniques known in the art, such as trypan blue exclusion assay,fluorescein diacetate uptake assay, propidium iodide uptake assay (toassess viability); and thymidine uptake assay, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cellproliferation assay (to assess proliferation). Longevity may bedetermined by methods well known in the art, such as by determining themaximum number of population doublings in an extended culture.

In one embodiment of the in vitro method, a colony forming unit assayusing placental stem cells and cord blood-derived stem cells isperformed as follows. Fresh or thawed HLA/donor matched placentalperfusate and cord blood units are obtained, and the number of totalnucleated cells in each is determined with a hemacytometer. Where thawedunits are used, cord blood samples can be hetastarch-separated, andplacental perfusate units are preferably Ficoll-separated. Small samplesof nucleated cells from each source are seeded together in suspension intwo or more ratios in a co-culture, and expanded. The co-culture can beperformed in, e.g., triplicate for one or more ratios of placental stemcells to stem cells from a second source, in, for example, 35 mm dishesin an appropriate cell culture medium (e.g., RPMI 1640 mediumsupplemented with 2-10% fetal calf serum and, optionally, 1% Stemspancytokine cocktail; Methocult GF⁺ H4435 medium, etc.). Hematopoietic stemcells may be expanded in culture medium comprising GM-CSF, IL-3, IL-6,SCF and flt-3 ligand.

The container used for the co-culture assay is preferably appropriatefor tissue culture of stem cells. For example, co-cultures may beperformed in glass or plastic Petri dishes, 16-well plates, 32-wellplates, 96-well plates, 128-well plates, and the like. Typically, thetotal number of nucleated cells from each source in each co-culturevaries from 1×10⁴ to 1×10⁶. Cells may also be co-cultured in amicropatterned configuration. See U.S. Pat. No. 6,221,663.

When determining the ratio of placental stem cells to stem cells from asecond source in a cell population that comprises a number of placentalstem cells and stem cells from a second source, the preferred ratio isany ratio that generates more colony forming units than that generatedby said number of placental stem cells or said number of stem cells froma second source under the same conditions. More preferably, the ratio isa ratio that generates a higher number of colony-forming units than allother ratios tested. Statistical significance between ratios tested isdesirable, but not necessary. The higher number of colony-forming unitsmay be attributable to, or be derived from, both placental stem cellsand stem cells from a second source; from predominantly or only theplacental stem cells; or predominantly or only the stem cells from asecond source.

The combined stem cell population is cultured for a time sufficient forcolony forming units to form, typically 10-20 days. Cell culture duringexpansion follows standard protocols known in the art of stem orprogenitor cell culture, and includes, for example, daily or semi-dailychanges of medium; culture at about 37° C. at 5% CO₂ in a humidifiedincubator, and the like. After 10-20 days, the number and morphology ofcolony forming units in the co-culture is determined (e.g., forhematopoietic stem cells, the number of CFU-GM, CFU-L, CFU-M, CFU-G,CFU-DC, CFU-GEMM, CFU-E).

In a specific example of the co-culture assay, nucleated cells fromplacenta perfusate, and nucleated cells from cord blood are combined aratio of 1:1, 1:3 and 3:1 (where 1 equals, e.g., 1×10⁵ cells) inMethocult GF⁺ H4435 medium. The co-culture is then expanded in tissueculture for about 14 days. The morphology of the co-cultured cells, andthe number of colony forming units, is determined. The ratio of thenucleated cell samples from the two sources that provides the highestnumber of colony-forming units is designated an optimum ratio, and thetwo units, or stem and/or progenitor cells from one or both of theunits, are combined in the optimum ratio for administration to arecipient in need of a stem cell transplant. Such an optimum ratioprovides superior engraftment in vivo over the administration of eitherunit, or stem and/or progenitor cells from either unit, alone, whereequivalent numbers of cells are administered.

The placental stem cells and stem cells from a second source arecontacted with each other during the co-culturing, either directly orindirectly. At a minimum, this comprises contacting one of the types ofstem cells with culture medium in which the other type of stem cell hascultured for a period of time, e.g., contacting one of the types of stemcells with medium that has been conditioned by the other type of stemcell. For example, the placental stem cells, and stem cells from asecond source may be cultured together in the same physical space duringculture for colony-forming unit formation, e.g., in the same culturedish or well in a multi-well plate. The placental stem cells and stemcells from a second source may also be contacted with each other byculturing in separate physical spaces, but in common culture medium(e.g., separated by a membrane, or in two wells of a multiwell platewherein culture medium may move actively or passively between the wells,but cells cannot mix). In another embodiment, placental stem cells andstem cells from a second source may be cultured in separate physicalspaces with no common culture medium, and the stem cells brought intocontact with each other by an exchange of part or all of the culturemedium from one stem cell culture with that of the other. In anotherembodiment, the cells in the co-culture are cultured in a manner thatphysically separates the cells, but allows biomolecules to diffusebetween the two cultures. See, e.g., U.S. Pat. No. 5,665,596 “Device forCell Co-culture and Method for Its Use in Culturing Cells”. Where thestem cell cultures are separate, the number of colony-forming units inthe separate, paired cultures is totaled for each replicate of ratio,and an optimum ratio determined, as above.

In another embodiment of the method, a bioactive molecule is added tothe placental stem cells and stem cells from a second source during theassay, and a ratio of placental stem cells to stem cells from a secondsource is identified that, for a total number of cells, results in morecolony-forming units, or enhanced engraftment, compared to a number ofplacental stem cells or stem cells from a second source, equivalent tosaid total number of cells in said combination, alone. Such a bioactivemolecule may be a small organic molecule of less than 50 kDa, 30 kDa, 20kDa, 10 kDa, 5 kDa, 3 kDa, 2 kDa, 1 kDa, 500 Da, 300 Da, 200 Da, 100 Daor smaller. In a specific embodiment, said small organic molecule issynthetic or non-natural, that is, not derived from a natural source. Inanother specific embodiment, said bioactive molecule is a cytokine orgrowth factor. Bioactive molecules that can be added to the co-cultureinclude differentiation-inducing agents such as, but are not limited to,Ca²⁺, EGF, α-FGF, β-FGF, PDGF, keratinocyte growth factor (KGF), TGF-β,cytokines (e.g., IL-1α, IL-1β, IFN-γ, TFN), retinoic acid, transferrin,hormones (e.g., androgen, estrogen, insulin, prolactin,triiodothyronine, hydrocortisone, dexamethasone), sodium butyrate, TPA,DMSO, NMF, DMF, matrix elements (e.g., collagen, laminin, heparansulfate, MATRIGEL™), or combinations thereof. Bioactive molecules thatare differentiation suppressants may also be added, such as, but notlimited to, human Delta-1 and human Serrate-1 polypeptides (see, Sakanoet al., U.S. Pat. No. 6,337,387 entitled “Differentiation-suppressivepolypeptide”, issued Jan. 8, 2002), leukemia inhibitory factor (LIF),and stem cell factor.

Where a bioactive molecule is added to the co-culture, the co-cultureassay may be used to identify a positive effector of engraftment. In oneembodiment, therefore, the invention provides a method of identifying abioactive molecule that is a positive effector of engraftment comprisingcontacting a combined stem cell population with said bioactive molecule,wherein said bioactive molecule is identified as a positive effector ofengraftment if engraftment by said combined stem cell population isdetectably enhanced compared to engraftment by a combined stem cellpopulation not contacted with said bioactive molecule. In anotherembodiment, the invention provides a method of identifying a positiveeffector of engraftment comprising combining placental stem cells andstem cells from a second source in vitro in one or more ratios in thepresence of said bioactive molecule; culturing said placental stem cellsand stem cells from a second source for a time sufficient for colonyforming units to form; determining the number of colony-forming unitsfor each of said one or more ratios; and determining, for at least oneof said one or more ratios, whether the number of colony forming unitsin the presence of said bioactive molecule is greater than the number ofcolony forming units in the absence of said bioactive molecule, and, ifso, identifying said bioactive molecule as a positive effector ofengraftment.

The in vitro assay may be performed on any placental stem cellpopulation and stem cell population from a second source to determine anoptimum ratio for engraftment. In this aspect, the in vitro co-cultureassay can be used as a standard, routine procedure to characterize stemcell populations prior to transplantation.

5.1.2 In Vivo Assay

The results of the above in vitro assay may be confirmed using an invivo engraftment assay. The in vivo assay may also be performed in theabsence of the in vitro assay to determine an optimum ratio of placentalstem cells, and stem cells from a second source, to maximizeengraftment.

In one embodiment of the in vivo assay, placental stem cells and stemcells from a second source are transplanted into a plurality of modelanimals and given sufficient time to engraft (typically 6-10 weeks). Theanimals are subsequently sacrificed, and the degree of engraftment ineach animal is determined for at least one tissue. Thus, in oneembodiment, the invention provides a method of identifying a ratio ofplacental stem cells and stem cells or progenitor cells from a secondsource for engraftment into a recipient, comprising identifying a ratioof placental stem cells to stem cells from a second source in a totalnumber of cells that, when transplanted into an animal, results inenhanced engraftment compared to transplantation of a number ofplacental stem cells or stem cells from a second source, equivalent tothe number of cells in said total number of cells, alone. In anotherembodiment, said identifying a ratio of placental stem cells to stemcells from a second source comprises transplanting a number of placentalstem cells and stem cells from a second source in a plurality ofanimals, in a plurality of ratios; determining the number of engraftedcells in at least one tissue of said animals for each of said pluralityof ratios; and identifying the ratio in said plurality of ratios thatyields the highest number of engrafted cells.

As in the in vitro assay, the placental stem cells can be placental stemcells obtained by any means or present in any usable form. For example,the placental stem cells may be contained in placental perfusate, or maybe contained within isolated total nucleated cells from the placentalperfusate, or may be a population of stem cells isolated from the totalnucleated cells, or may be placental stem cells contained withinenzyme-digested placental tissue, or may be placental stem cellsisolated from enzyme-digested placental tissue, or may be placental stemcells that have been expanded and/or passaged in culture, etc.

Any standard model animal may be used in the in vivo co-culture assay.Preferably, the model animal is one in which engraftment of xenograftsmay be readily accomplished. Small mammals such as standard laboratoryrodents such as mice and rats are preferred because they require feweradministered stem cells to show engraftment. It is highly preferablethat the model animal be immune-compromised. Animal models that may beused in the in vivo assay include, but are not limited to, NOD/SCID(non-obese diabetic/severe combined immune deficiency) mice (see Hoganet al., Blood 90(1):85-96 (1997)); beige/nude/x-linked immunodeficiency(BNX) mice (see, e.g., Kamal-Reid et al., Science 242:1706 (1988)); SCIDmice (see, e.g., Kamal-Reid et al., Science 246:1597 (1989). Engraftmentmay be accomplished in other animal models, such as sheep fetuses (see,e.g., Shimizu et al., Blood 91(10):3688-3692 (1998); Zanjani et al.,Int'l J. Hematol 63(3): 179-182 (1996)).

The determination of the number of engrafted cells in tissues from therecipient animal may be accomplished by any means known in the art. Forexample, detection of engrafted cells may be accomplished by detectionof engrafted cell-specific nucleic acids, e.g., by the polymerase chainreaction, or by detection of proteins specific for engrafted cells,e.g., by immunohistochemistry. Identification of engraftment in vivo maybe determined through the use of a sample, e.g., biopsy specimen, takenat one or more locations on, and at one or more post-transplantationtimes from, a recipient.

In one embodiment, demonstration of engraftment of placental stem cellsand/or cord blood-derived stem cells can be accomplished by taking abiopsy (e.g., bone marrow aspirate or peripheral blood sample) andperforming PCR to determine whether any non-recipient genetic markersare present, which would indicate engraftment. In another embodiment,identification of engrafted cells is accomplished by selection of one ormore antibodies that recognize markers expressed by the engrafted cells.In a specific embodiment, the engrafted cells are human, and the one ormore antibodies specifically recognize one or more human cell markers.Antibodies can be used to detect the markers by any art-accepted method,e.g., immunohistochemical methods. For example, determination of thepresence of a cell surface marker can comprise sacrifice of a non-humanhost animal, obtaining a desired tissue, fixing and embedding the tissuein paraffin or a similar matrix; thin sectioning the tissue, optionallyfollowed by staining; and contacting the tissue with one or moreantibodies that recognize the marker. In the same manner, one may useantibodies that recognize markers expressed by cells into which theengrafted stem cells can differentiate. For example, placental stemcells or cord blood-derived stem cells differentiate into cells thatexpress CD45 and vimentin; thus, antibodies to CD45 and vimentin may beused to determine the number of engrafting, and differentiating, stemcells. Antibodies that recognize, e.g., human cell surface markers inpreference to host cell markers, e.g., mouse cell surface markers, arewell-known in the art.

In a non-limiting example of the in vivo method, a plurality of modelanimals, e.g., a plurality of mice of the species Mus musculus, aretransplanted with human placental stem cells and, e.g., human nucleatedcells isolated from cord blood, including hematopoietic stem cells, in aplurality of ratios. After several days to several weeks (i.e.,sufficient time to allow engraftment), the host animals are sacrificed,and tissues (e.g., spleen, lung, etc.) are examined to determine theapproximate number of human cells that have engrafted, as evidenced bythe number of cells staining for CD45 and/or vimentin. CD45 is a markerspecific for leukocytes, including T- and B-lymphocytes, granulocytes,monocytes and macrophages. Certain CD45 antibodies, such as clone T29/33(BioDesign, Saco, Me.), do not cross-react with mouse antigens. Vimentinis a marker for mesenchymal cells, such as fibroblasts, smooth musclecells, lipocytes, Schwann cells, vascular endothelial cells, and thelike. Certain vimentin antibodies, such as clone V9 (BioDesign, Saco,Me.), do not cross-react with mouse antigens. Staining with antibodiesto these two markers, therefore, can establish generally the extent ofengraftment of placental stem cells, and stem cells from a secondsource, in a variety of tissues. This example is not limiting; differentantibodies may be used to determine the extent of engraftment of othercell types. In a long-term engraftment model, bone marrow cells isolatedfrom a primary engrafted animal, e.g., a mouse, can be transplanted intoa second engraftment model animal. Assays for secondary engraftment areas listed above and include methods well known to those of skill in theart.

5.2 Combined Stem Cell Populations

The invention further provides combined stem cell compositionscomprising placental stem cells, e.g., cells from placental perfusate,e.g., nucleated cells from placental perfusate, comprising placentalstem cells and stem cells from a second source that, for a particularnumber of cells, results in a greater number of colony-forming units ina colony-forming unit assay, or enhanced engraftment in a transplantrecipient, than the number of either placental stem cells or stem cellsfrom a second source, alone. Combined stem cell populations identifiedby the above methods represent engraftment-enhanced combinations of stemcells based on the characteristics of the stem cell sources, that is,the number of engraftable cells contained in, e.g., a unit of placentaperfusate, a unit of cord blood, etc.

Thus, in one embodiment, the invention encompasses a combined stem cellcomposition comprising a number of placental stem cells and stem cellsfrom a second source in a ratio, wherein the stem cells from thecomposition show improved engraftment compared to a number of either theplacental stem cells or the stem cells from a second source, equivalentto the number of cells in said composition, alone. In a specificembodiment, the ratio is identified by combining placental stem cellsand stem cells from a second source in vitro in a plurality of ratiosfor a time and under conditions sufficient to allow the formation ofcolony-forming units; and identifying a ratio in said plurality ofratios that yields the highest number of colony forming units. In a morespecific embodiment, said stem or progenitor cells from a second sourceare cord blood stem or progenitor cells, bone marrow stem or progenitorcells, hematopoietic stem or progenitor cells, or mesenchymal stem orprogenitor cells. In another more specific embodiment, said stem cellsor progenitor cells from a second source are hematopoietic progenitorcells. In an even more specific embodiment, said hematopoietic stemcells are cord blood hematopoietic stem cells. In another even morespecific embodiment, said hematopoietic cells are CD34⁺ cells.

In another more specific embodiment, said placental stem cells compriseCD34⁺ cells, for example, CD34⁺CD38⁺ cells and/or CD34⁺CD38⁻ cells. In aspecific embodiment, said CD34⁺CD38⁻ cells comprise CD34⁺CD38⁻lin⁻ stemcells. In another specific embodiment, said CD34+ placental stem cellscomprise cells that are ALDH+, that is, CD34⁺, ALDH⁺ placental stemcells.

In another more specific embodiment, said placental stem cells areOCT-4⁺ or ABC-p⁺. In another more specific embodiment, said placentalstem cells comprise cells that are OCT4⁺ABC-p⁺. In another more specificembodiment, said placental stem cells comprise cells that are CD34⁺ andcells that are OCT4⁺ABC-p⁺. In another more specific embodiment, saidplacental stem cells are contained within placental perfusatesubstantially lacking red blood cells and cellular debris. In anothermore specific embodiment, said composition comprises placental stemcells isolated from placental perfusate.

In another embodiment, placental stem cells comprise cells that expressone or more of markers CD10, CD29, CD44, CD54, CD90, CD73 or CD105, andlack one or more of markers CD34, CD38, CD45, SSEA3 and SSEA4. Inanother embodiment, placental stem cells comprise cells that arepositive for CD10, CD29, CD44, CD54, CD90, CD73 or CD105, and negativefor CD34, CD38, CD45, SSEA3 and SSEA4. In another embodiment, placentalstem cells comprise cells that comprise one or more of markers CD10,CD29, CD44, CD54, CD90, CD73 and CD105, and lack one or more of markersCD34, CD38, CD45, SSEA3 and SSEA4. In another embodiment, placental stemcells comprise cells that are positive for CD10, CD29, CD44, CD54, CD90,CD73 and CD105, and negative for CD34, CD38, CD45, SSEA3 and SSEA4. Inanother embodiment, said placental stem cells comprise CD34⁻ cells. In aspecific embodiment, said placental stem cells are CD34⁻CD38⁻ placentalstem cells. In another embodiment, said placental stem cells comprisecells that are positive for at least one of CD10, CD29, CD33, CD44,CD73, CD105, CD117, and CD133, and negative for at least one of CD34 orCD45. In another embodiment, said placental stem cells comprise cellsthat are positive for CD10, CD29, CD33, CD44, CD73, CD105, CD117, andCD133, and negative for CD34 or CD45. In a more specific embodiment,said placental stem cells comprise cells that are HLA-ABC⁺. In a morespecific embodiment, said placental stem cells comprise cells that areHLA-ABC⁻. In a more specific embodiment, said placental stem cellscomprise cells that are HLA-DR⁺. In a more specific embodiment, saidplacental stem cells comprise cells that are HLA-DR⁻. In anotherspecific embodiment, said placental stem cells comprise cells that areCD200⁺ or HLA-G⁺. In another specific embodiment, the placental stemcells comprise cells that are CD200⁺ and HLA-G⁺. In another specificembodiment, the placental stem cells comprise cells that are CD73⁺,CD105⁺ and CD200⁺. In another specific embodiment, the placental stemcells comprise cells that are CD200⁺ and OCT-4⁺. In another specificembodiment, the placental stem cells comprise cells that are CD73⁺,CD105⁺ and facilitate the formation of embryoid-like bodies in apopulation of isolated placental cells comprising said stem cells, whensaid population is cultured under conditions that allow the formation ofembryoid-like bodies. In another specific embodiment, the placental stemcells comprise cells that are CD73⁺, CD105⁺ and HLA-G⁺. In anotherspecific embodiment, the placental stem cells comprise cells that areOCT-4⁺ and facilitate the formation of embryoid-like bodies in apopulation of isolated placental cells comprising said stem cells, whensaid population is cultured under conditions that allow the formation ofembryoid-like bodies.

In another embodiment, said stem cells from a second source are stemcells derived from cord blood.

In the combined stem cell populations of the invention, the placentalstem cells and the stem cells from a second source may beidentically-HLA-matched, that is, they may be derived from the sameindividual. In another embodiment, the placental stem cells and the stemcells from a second source may be HLA-mismatched, that is, they may bederived from different individuals. For combined stem cell populationscomprising cord blood or cord blood-derived stem cells, the combinationmay also comprise stem cells that are either HLA-matched, partiallyHLA-matched, or HLA-mismatched to an intended recipient. For combinedstem cell populations comprising non-cord blood stem cells, it ispreferred that at least the stem cells from a second source beHLA-matched or partially HLA-matched to the intended recipient.

In various embodiments, the ratio of placental stem cells to stem cellsfrom a second source can be about 100,000,000:1, 50,000,000:1,20,000,000:1, 10,000,000:1, 5,000,000:1, 2,000,000:1, 1,000,000:1,500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1, 10,000:1, 5,000:1,2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1;1:2; 1:5; 1:10; 1:100; 1:200; 1:500; 1:1,000; 1:2,000; 1:5,000;1:10,000; 1:20,000; 1:50,000; 1:100,000; 1:500,000; 1:1,000,000;1:2,000,000; 1:5,000,000; 1:10,000,000; 1:20,000,000; 1:50,000,000; orabout 1:100,000,000, comparing numbers of total nucleated cells in eachpopulation, or comparing total numbers of stem cells in each population.In a preferred embodiment, the ratio of placental stem cells to stemcells from a second source can be about 1:10 to about 10:1. In otherpreferred embodiments, the ratio of placental stem cells to stem cellsfrom a second source can be about 3:1 to about 1:3.

The combined stem cell populations of the invention can comprise atherapeutically-effective amount of placental stem cells, stem cellsfrom a second source, or both. The combined stem cell populations of theinvention, and pharmaceutical compositions comprising a combined stemcell population, can comprise at least 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵,1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰or 1×10¹¹ placental stem cells, stem cells from a second source, orboth, or no more than 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷,5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰ or 1×10¹¹ placentalstem cells, stem cells from a second source, or both.

In other embodiments, said combined stem cell population improvesengraftment in an individual in need of stem cells at least, or at, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21days post-transplant. In another more specific embodiment, said combinedstem cell population improves engraftment in an individual in need ofstem cells at least, or at, more than 21 days post-transplant. Inspecific embodiments, said combined stem cell population improvesengraftment in an individual in need of stem cells at least, or at, morethan 25, 30, 35, 40, 45, 50, 55 weeks, or 1 year or longerpost-transplant.

The combined stem cell populations of the invention can be preserved,for example, cryopreserved for later use. Methods for cryopreservationof cells, such as stem cells, are well known in the art, for example,cryopreservation using the methods of Boyse et al. (U.S. Pat. No.5,192,553, issued Mar. 9, 1993) or Hu et al. (WO 00/73421, publishedDec. 7, 2000). Placenta-derived stem cells, and stem cells from a secondsource, which make up a combined stem cell population, can be combinedprior to cryopreservation, or can be cryopreserved separately, andcombined in the appropriate ratio upon thawing, e.g., within hours ofuse.

The combined stem cell populations of the invention can be prepared in aform that is easily administrable to an individual. For example, acombined stem cell population can be contained within a containersuitable for medical use. Such a container can be, for example, asterile plastic bag, flask, jar, or other container from which thecombined stem cell population can be easily dispensed. Preferably, thecontainer is a container that allows, or facilitates, intravenousadministration of a combined stem cell population. The container, e.g.,bag, can hold the placenta-derived stem cells and stem cells from asecond source together, e.g., as a mixed cell population, or can holdthe two stem cell populations separately. In the latter embodiment, thebag preferably comprises multiple lumens or compartments that areinterconnected to allow mixing of the placenta-derived stem cells andstem cells from a second source prior to, or during, administration. Thecontainer is preferably one that allows for cryopreservation of thecombined stem cell population. The combined stem cell population in saidcontainer can comprise placenta-derived stem cells, stem cells from asecond source, or both, that have been passaged at least, or at most, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20times, or 25, 30, 35, 40 or more times.

The invention also provides for combined stem cell populations thatcomprise, e.g., that are stored or maintained as, separate stem cellpopulations, e.g., a population of placenta-derived stem cells and apopulation of stem cells from a second source, in combination withinformation on combining the two populations in an appropriate ratioprior to use, e.g., prior to administration to an individual in need ofstem cells. In this embodiment, a combined stem cell population wouldcomprise a population of placenta-derived stem cells in a firstcontainer, a population of stem cells from a second source in a secondcontainer, and instructions for combining the two populations eitherbefore or during administration to an individual in need of stem cells.

Thus, in one embodiment, the invention provides a composition comprisinga combined stem cell population in a container, wherein said combinedstem cell population comprises placenta-derived stem cells and stemcells from a second source. In a specific embodiment, the container is abag, flask, or jar. In a more specific embodiment, said placenta-derivedstem cells and said stem cells from a second source are containedtogether in said bag. In another more specific embodiment, saidplacenta-derived stem cells and said stem cells from a second source arecontained separately within said bag. In another specific embodiment,the composition comprises one or more compounds that facilitatecryopreservation of the combined stem cell population. In anotherspecific embodiment, said combined stem cell population is containedwithin a physiologically-acceptable aqueous solution. In a more specificembodiment, said physiologically-acceptable aqueous solution is a 0.9%NaCl solution. In another more specific embodiment, said bag is asterile plastic bag. In a more specific embodiment, said bag allows orfacilitates intravenous administration of said combined stem cellpopulation. In another specific embodiment, said combined stem cellpopulation comprises placental cells that are HLA-matched to said stemcells from a second source. In another specific embodiment, saidcombined stem cell population comprises placental cells that are atleast partially HLA-mismatched to said stem cells from a second source.In another specific embodiment, said placenta-derived stem cells arederived from a plurality of donors. In another specific embodiment, saidstem cells from a second source are derived from a plurality of donors.

Combined stem cell populations can be cultured for a period of timeprior to administration to an individual. For example, in oneembodiment, the stem cells in a combined stem cell population can becultured in medium comprising Notch agonist, e.g., a deletion form of aNotch protein consisting essentially of the intracellular domain of theNotch protein, or a Delta protein. See U.S. 2004/0067583

5.3 Pharmaceutical Compositions

The present invention encompasses pharmaceutical compositions thatcomprise combined stem cell populations of the invention, and apharmaceutically-acceptable carrier.

In accordance with this embodiment, the combined stem cell populationsof the invention may be formulated as an injectable (e.g., WO 96/39101,incorporated herein by reference in its entirety). In anotherembodiment, the combined stem cell populations of the present inventionmay be formulated using polymerizable or cross linking hydrogels asdescribed, e.g., in U.S. Pat. Nos. 5,709,854; 5,516,532; 5,654,381.

In another embodiment, the invention provides for the maintenance ofeach stem cell population of the combined stem cell populations, priorto administration to an individual, as separate pharmaceuticalcompositions to be administered sequentially or jointly to create thecombined stem cell population in vivo. Each component may be storedand/or used in a separate container, e.g., one bag (e.g., blood storagebag from Baxter, Becton-Dickinson, Medcep, National Hospital Products,Terumo, etc.) or separate syringe, which contains a single type of cellor cell population. In a specific embodiment, cord blood, or cordblood-derived nucleated or stem cells, are contained in one bag, andplacental perfusate, or placental stem cells from placental perfusate,are contained in a second bag.

A population of placental stem cells can be enriched. In a specificembodiment, a population of cells comprising placental stem cells isenriched by removal of red blood cells and/or granulocytes according tostandard methods, so that the remaining population of nucleated cells isenriched for placental stem cells relative to other cell types inplacental perfusate. Such an enriched population of placental stem cellsmay be used unfrozen, or may be frozen for later use. If the populationof cells is to be frozen, a standard cryopreservative (e.g., DMSO,glycerol, EPILIFE™ Cell Freezing Medium (Cascade Biologics)) is added tothe enriched population of cells before it is frozen.

The pharmaceutical compositions of the invention may comprise one ormore agents that induce cell differentiation. In certain embodiments, anagent that induces differentiation includes, but is not limited to,Ca²⁺, EGF, α-FGF, β-FGF, PDGF, keratinocyte growth factor (KGF), TGF-β,cytokines (e.g., IL-1α, IL-1β, IFN-γ, TFN), retinoic acid, transferrin,hormones (e.g., androgen, estrogen, insulin, prolactin,triiodothyroxine, hydrocortisone, dexamethasone), sodium butyrate, TPA,DMSO, NMF, DMF, matrix elements (e.g., collagen, laminin, heparansulfate, MATRIGEL™), or combinations thereof.

In another embodiment, the pharmaceutical composition of the inventionmay comprise one or more agents that suppress cellular differentiation.In certain embodiments, an agent that suppresses differentiationincludes, but is not limited to, human Delta-1 and human Serrate-1polypeptides (see, Sakano et al., U.S. Pat. No. 6,337,387), leukemiainhibitory factor (LIF), stem cell factor, or combinations thereof.

The pharmaceutical compositions of the present invention may be treatedprior to administration to an individual with a compound that modulatesthe activity of TNF-α. Such compounds are disclosed in detail in, e.g.,U.S. Application Publication No. 2003/0235909, which disclosure isincorporated herein in its entirety. Preferred compounds are referred toas IMiDs (immunomodulatory compounds) and SelCIDs (Selective CytokineInhibitory Drugs), and particularly preferred compounds are availableunder the trade names ACTIMID™, REVIMID™ and REVLIMID™.

5.4 Methods of Transplanting Stem Cells

5.4.1 Transplantation Methods

The above method of identifying combined stem cell populations (seeSection 5.1) may be performed on paired units of, for example, placentalperfusate or placental stem cells, and stem cells from a second source,e.g., cord blood, cord blood stem cells, and the like, to producecombined stem cell populations for the treatment of an individual inneed of stem cells. In one embodiment, the individual is contacted withone or more combined stem cell populations. In a specific embodiment,said contacting is the introduction, e.g., transplantation, of saidcombined stem cell population into said individual. Thus, the method ofproducing combined stem cell populations may be performed as a firststep in a procedure for introducing stem cells into any individualneeding stem cells. Such a procedure can comprise use of pharmaceuticalcompositions comprising the combined stem cell populations, as describedabove.

In a specific embodiment, a population of placental stem cells of theinvention is combined with a population of stem cells from a secondsource prior to administration to an individual in need thereof in aratio that provides improved or enhanced engraftment over a number ofsaid placental stem cells or said stem cells from a second source,equivalent to said total number of cells, alone. In another specificembodiment, a population of placental stem cells of the invention iscombined with a population of stem cells from a second source during, orsimultaneously with, administration to a patient in need thereof, in anoptimum ratio, wherein said ratio is identified by identifying a ratioof placental stem cells to stem cells from a second source, in aplurality of ratios, that yields the highest number of saidcolony-forming units when said placental stem cells and stem cells froma second source are cultured for a time and under conditions sufficientto allow the formation of colony-forming units. In another specificembodiment, a population of placental stem cells of the invention and apopulation of umbilical cord blood cells are administered sequentiallyto a patient in need thereof to a final optimum ratio. In oneembodiment, the population of placental stem cells is administered firstand the population of stem cells from a second source is administeredsecond. In another embodiment, the population of stem cells from asecond source is administered first and the population of placental stemcells is administered second.

In a specific embodiment, said combined stem cell population iscontained within one bag or container. In another embodiment, theinvention provides for use in transplantation of a population ofplacental stem cells, and stem cells from a second source, that arecontained within separate bags or containers. In certain embodiments,stem cell populations contained in two bags may be mixed prior, inparticular immediately prior, to or at the time of administration to apatient in need thereof. In other embodiments, the contents of each bagmay be administered separately to a patient, wherein two cellpopulations are used adjunctively in vivo.

Combined populations of placental stem cells, and stem cells from asecond source, e.g., cord blood-derived stem or progenitor cells, orcord blood, including banked or cryopreserved cord blood may be mixed,prior to transplantation, by any medically-acceptable means. In oneembodiment, the two populations are physically mixed. In anotherembodiment of the method, said placental stem cells and stem cells froma second source are mixed immediately prior to (i.e., within 1, 2, 3, 4,5, 7, 10 minutes of) administration to said individual. In anotherembodiment, said placental stem cells and stem cells from a secondsource are mixed at a point in time more than five minutes prior toadministration to said individual. In another embodiment of the method,the placental stem cells, and/or stem cells from a second source, arecryopreserved and thawed prior to administration to said individual. Inanother embodiment, said placental stem cells and stem cells from asecond source are mixed to form a combined stem cell population at apoint in time more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours prior to administrationto said individual, wherein either or both of said placental stem cellsand stem cells from a second source have been cryopreserved and thawedprior to said administration. In another embodiment, the combined stemcell populations may be administered more than once.

In another embodiment, the stem cells contained within the combined stemcell population are preconditioned prior to transplantation. In apreferred embodiment, preconditioning comprises storing the cells in agas-permeable container generally for a period of time at about −5° C.to about 23° C., about 0° C. to about 10° C., or preferably about 4° C.to about 5° C. The cells may be stored between 18 hours and 21 days,between 48 hours and 10 days, preferably between 3-5 days. The cells maybe cryopreserved prior to preconditioning or, may be preconditionedimmediately prior to administration.

Once an appropriate ratio of placental stem cells to stem cells from asecond source is established, either or both of the placental stemcells, or stem cells from a second source, may be differentiated priorto introduction to an individual in need of stem cells. For example, forintroduction for the purpose of hematopoietic engraftment, the stemcells may be differentiated to cells in the hematopoietic lineage. Thecombination of stem cells and differentiated cells, or combination ofcells differentiated from both sources of stem cells, is encompassedwithin the term “combined stem cell population.” Thus, the inventionprovides a method of introducing stem cells into an individualcomprising determining a ratio of placental stem cells and stem cellsfrom a second source in a total number of cells, wherein the ratioimproves engraftment as compared to introduction of a number ofplacental stem cells or stem cells from a second source, equivalent tosaid total number of cells, alone; differentiating one or both of saidplacental stem cells or stem cells from a second source into cells ofanother cell type; and introducing said stem cells and/or differentiatedcells to an individual. In certain embodiments of the invention, themethod of transplantation of combined stem cell populations comprises(a) induction of differentiation of placental stem cells, (b) mixing theplacental stem cells with a population of stem cells from a secondsource, e.g., cord blood stem cells, to form a combined cell population,and (c) administration of the combined cell population to an individualin need thereof. In another embodiment the method of transplantationcomprises (a) induction of differentiation of stem cells from a secondsource; (b) mixing the differentiated cells with placental stem cells toform a combined cell population; and (c) administration of the combinedcell population to an individual in need thereof. In another embodimentof the invention, the method of transplantation of combined stem cellpopulations comprises (a) mixing placental stem cells with a populationof cord blood cells; (b) induction of differentiation of the mixture ofthe cord blood cells and placental stem cells and (c) administration ofthe mixture to a patient in need thereof.

The combined stem cell populations of the invention may be transplantedinto a patient in any pharmaceutically or medically acceptable manner,including by injection, e.g., intravenous injection, intramuscularinjection, intraperitoneal injection, intraocular injection, directinjection into a particular tissue, transfusion, etc. For example,combined stem cell populations, e.g., placental stem cells incombination with cord blood-derived stem cells) may be transplanted byintravenous infusion. In another embodiment, a combined stem cellpopulation comprising placental stem cells and cardiac stem cells, insuspension, may be injected directly into cardiac tissue, e.g., anischemic area in a heart. The combined stem cell populations maycomprise, or be suspended in, any pharmaceutically-acceptable carrier.The combined stem cell populations may be carried, stored, ortransported in any pharmaceutically or medically acceptable container,for example, a blood bag, transfer bag, plastic tube or vial.

After transplantation, engraftment in a human recipient may be assessedusing, e.g., nucleic acid or protein detection or analytical methods.For example, the polymerase chain reaction (PCR), STR, SSCP, RFLPanalysis, AFLP analysis, and the like, may be used to identify engraftedcell-specific nucleotide sequences in a tissue sample from therecipient. Such nucleic acid detection and analysis methods arewell-known in the art. In one embodiment, engraftment may be determinedby the appearance of engrafted cell-specific nucleic acids in a tissuesample from a recipient, which are distinguishable from background. Thetissue sample analyzed may be, for example, a biopsy (e.g., bone marrowaspirate) or a blood sample.

In one embodiment, a sample of peripheral blood is taken from a patientimmediately prior to a medical procedure, e.g., myeloablation. After theprocedure, a combined stem cell population of the invention isadministered to the patient. At least once post-administration, a secondsample of peripheral blood is taken. An STR profile is obtained for bothsamples, e.g., using PCR primers for markers (alleles) available from,e.g., LabCorp (Laboratory Corporation of America). A difference in thenumber or characteristics of the markers (alleles) post-administrationindicates that engraftment has taken place.

Engraftment can also be demonstrated by detection of re-emergence ofneutrophils.

In another example, engrafted cell-specific markers may be detected in atissue sample from the recipient using antibodies directed to markersspecific to either the transplanted stem cells, or cells into which thetransplanted stem cells would be expected to differentiate. In oneembodiment, engraftment of a combination of placental stem cells andcord blood-derived stem cells may be assessed by FACS analysis todetermine the presence of CD45⁺, CD19⁺, CD33⁺, CD7⁺ and/or CD3⁺ cells byadding the appropriate antibody and allowing binding; washing (e.g.,with PBS); fixing the cells (e.g., with 1% paraformaldehyde); andanalyzing on an appropriate FACS apparatus (e.g., a FACSCalibur flowcytometer (Becton Dickinson)). In another embodiment, engraftment of acombination of placental stem cells and cord blood-derived stem cellsmay be assessed by FACS analysis to determine the presence of CD200⁺ orHLA-G⁺ cells. Where placental stem cells and/or stem cells from a secondsource are from an individual of a different sex than a recipient, e.g.,male donor and female recipient, engraftment can be determined bydetection of sex-specific markers, e.g., Y-chromosome-specific markers.Placental stem cells and/or stem cells from a second source may also begenetically modified to express a unique marker or nucleic acid sequencethat facilitates identification, e.g., an RFLP marker, expression ofβ-galactosidase or green fluorescent protein, or the like.

The degree of engraftment may be assessed by any means known in the art.In one embodiment, the degree of engraftment is assessed by a gradingsystem as follows, which uses a thin section of fixed and antibody-boundtissue from the transplant recipient. In this example grading system,engraftment is graded as follows: 0=no positive cells (that is, no cellsbound by an antibody specific to an engrafted cell); 0.5=one or twopositive cells, perhaps positive, but difficult to differentiate frombackground or non-specific staining; 1=2-20 scattered positive cells;2=approximately 20-100 scattered or clustered positive cells throughoutthe tissue; 3=more than 100 positive cells comprising less than 50% ofthe tissue; 4=more than 50% of cells are positive. In specificembodiments, engraftment is determined where greater than 0.5%, 1%, 2%,3%, 4%, 5%, 7.5%, 10%, 15%, 20% or greater of the cells are positivelystained.

In another embodiment, the degree of engraftment is determined byanalysis of the gain of one or more biological functions carried out bythe engrafted cells. For example, where a recipient, who has undergonemyeloablative therapy, receives a transplant of a combined stem cellpopulation comprising placental stem cells and cord blood-derived stemcells, the degree of engraftment may be determined by the degree towhich normal hematopoiesis, blood cell populations and blood functionreturn to normal.

Where the combined stem cell population in whole or in part isHLA-mismatched to an intended recipient, it may be necessary to treatthe recipient to reduce immunological rejection of the donor cells.Methods for reducing immunological rejection are disclosed in, e.g.,U.S. Pat. Nos. 5,800,539 and 5,806,529, both of which are incorporatedherein by reference.

5.4.2 Dosages

Typically, a patient receiving a stem cell infusion, for example for abone marrow transplantation, receives one unit of nucleated cells, wherea unit is approximately 1×10⁹ nucleated cells (corresponding to 1-2×10⁶CD34⁺ stem cells). Transplantation of a combined stem cell populationinto an individual comprises, in various embodiments, transplantation ofat least one hundred thousand, 1 million, 10 million, 100-200 million, 1billion, 3 billion, 5 billion, 10 billion, 15 billion, 20 billion, 30billion, 40 billion, 50 billion or more, or, alternatively, 3, 5, 10,20, 30, 40, or 50 units or more, of total nucleated cells, from both theplacental stem cell population and the stem cell population from asecond source. Transplantation of a combined stem cell population intoan individual comprises, in other embodiments, transplantation of atleast 10-20 million, 100 million, 300 million, 500 million, 1 billion,1.5 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7billion, 8 billion, 9 billion, 10 billion or more stem cells. In anotherembodiment, the number of nucleated cells administered to an individualis at least five times the number of cells normally administered in abone marrow replacement. In another specific embodiment of the method,the number of nucleated cells administered to an individual is at leastten times the number of cells normally administered in a bone marrowreplacement. In another specific embodiment, the number of nucleatedcells administered to an individual is at least fifteen times the numberof cells normally administered in a bone marrow replacement. In anotherembodiment of the method, the total number of nucleated cells, whichincludes stem cells, administered to an individual is between 1-1000×10⁸per kilogram of body weight.

5.5 Methods of Treatment Using Combined Stem Cell Populations

The combined stem cell populations of the invention can be used to treatan individual in need of engraftable stem cells. Such an individual, forexample, may require a transplantation of stem cells to effecthematopoietic reconstitution. In various other embodiments, the combinedstem cell populations may be used to treat an individual having a bloodcancer, a lysosomal storage disease, an inflammatory disorder, or anautoimmune disorder. In other embodiments, the combined stem cellpopulations may be used to facilitate organ regeneration or repair, ormay be used as a transgene carrier.

Thus, in one embodiment, the invention provides a method of treating anindividual, comprising contacting (e.g., administering to) an individualwith a combined stem cell population of the invention. In anotherembodiment, the invention provides a method of treating an individualcomprising identifying a combined stem cell population, and contactingsaid individual with said combined stem cell population. In a specificembodiment, the combined stem cell populations comprise placental stemcells and stem cells from a second source in a ration, in a total numberof cells, that improves or enhances engraftment compared to a number ofplacental stem cells or stem cells from a second source, equivalent tosaid total number of cells, alone. In another embodiment, the inventionprovides a method of treating an individual, comprising introducing tosaid individual a composition comprising placental stem cells and stemcells from a second source in a ratio, wherein said ratio is selected byidentifying a ratio in a plurality of ratios of numbers of placentalstem cells to stem cells from a second source that, when cultured invitro for a time and under conditions sufficient to allow the formationof colony-forming units, produces the greatest number of colony formingunits, the numbers of cells in the colony-forming unit being equivalentin each condition, wherein said individual has a disease, disorder orcondition treatable with stem cells. In a specific embodiment, said stemcells from a second source are umbilical cord blood or placental bloodstem cells. In another specific embodiment, said stem cells from asecond source are hematopoietic stem cells. In another specificembodiment, said stem cells from a second source are bone marrow-derivedstem cells. In another specific embodiment, said treating isprophylactic. In another specific embodiment, said treating istherapeutic. In various embodiments, said disease, disorder or conditionis one of the diseases, disorders or conditions listed below. The listof diseases, disorders, and conditions provided herein is not intendedto be limiting.

One use of combined stem cell populations, particularly stem cellpopulations comprising placental stem cells and umbilical cord blood, orumbilical cord blood-derived stem cells, is hematopoietic reconstitutionin, e.g., patients who have undergone partial or complete myeloablativetherapy as part of an anticancer regimen. Typically bone marrow stemcells are transplanted to effect hematopoietic reconstitution, at adosage of approximately 1×10⁸ to 2×10⁸ bone marrow mononuclear cells perkilogram of patient weight must be infused for engraftment in a bonemarrow transplantation, or about 1-8×10⁶ CD34⁺ stem cells (i.e., about70 ml of marrow for a 70 kg donor). Hematopoietic reconstitution may beaccomplished by introduction to an individual of an equivalent number oftotal nucleated cells in a combined stem cell population comprising,e.g., placental stem cells and stem cells from a second source, e.g,placental blood or cord blood.

Placental stem cells and stem cells from a second source can be fully orpartially immunologically matched to a recipient, or can be from acompletely unrelated individual. In one embodiment, individualsreceiving a combined stem cell population receive ≧3.5×10⁷ totalnucleated cells (TNC), e.g., from umbilical cord blood, per kg bodyweight for 5/6 HLA matched cells, or ≧5.0×10⁷ total nucleated cells(TNC)/kg body weight for 4/6 HLA matched cells. Infusion of TNC, e.g.,from UCB, is followed, e.g., immediately, by an infusion of about 5 toabout 30×10⁶ TNC from placental perfusate per kg body weight. Anindividual can receive a single of such doses, or multiple such doses.

In one embodiment, therefore, combined stem cell populations comprisinghematopoietic stem cells can be used to treat patients having a bloodcancer, such as a lymphoma, leukemia (such as chronic or acutemyelogenous leukemia, acute lymphocytic leukemia, Hodgkin's disease,etc.), myelodysplasia, myelodysplastic syndrome, and the like. Inanother embodiment, the disease, disorder or condition is chronicgranulomatous disease.

Because hematopoietic reconstitution can be used in the treatment ofanemias, the present invention further encompasses the treatment of anindividual with a stem cell combination of the invention, wherein theindividual has an anemia or disorder of the blood hemoglobin. The anemiaor disorder may be natural (e.g., caused by genetics or disease), or maybe artificially-induced (e.g., by accidental or deliberate poisoning,chemotherapy, and the like). In another embodiment, the disease ordisorder is a marrow failure syndrome (e.g., aplastic anemia, Kostmannsyndrome, Diamond-Blackfan anemia, amegakaryocytic thrombocytopenia, andthe like), a bone marrow disorder or a hematopoietic disease ordisorder.

In another embodiment, the combined stem cell populations of theinvention can be introduced into a damaged organ for organ neogenesisand repair of injury in vivo. Such injury may be due to conditions anddisorders including, but not limited to, myocardial infarction, seizuredisorder, multiple sclerosis, stroke, hypotension, cardiac arrest,ischemia, inflammation, age-related loss of cognitive function, cerebralpalsy, neurodegenerative disease, Alzheimer's disease, Parkinson'sdisease, Leigh disease, AIDS dementia, memory loss, amyotrophic lateralsclerosis, ischemic renal disease, brain or spinal cord trauma,heart-lung bypass, glaucoma, retinal ischemia, or retinal trauma.

In other embodiments, the disease, disorder or condition treatable usingthe combined stem cell populations include, but are not limited tolysosomal storage diseases, such as Tay-Sachs, Niemann-Pick, Fabry's,Gaucher's disease (e.g., glucocerebrosidase deficiency), Hunter's, andHurler's syndromes, Maroteaux-Lamy syndrome, fucosidosis (fucosidasedeficiency), Batten disease (CLN3), as well as other gangliosidoses,mucopolysaccharidoses, and glycogenoses.

The combined stem cell populations can also be used to treat severecombined immunodeficiency disease, including, but not limited to,combined immunodeficiency disease (e.g., Wiskott-Aldrich syndrome,severe DiGeorge syndrome, and the like).

In other embodiments, combined stem cell populations may be used asautologous or heterologous transgene carriers in gene therapy tocorrect, for example, inborn errors of metabolism, adrenoleukodystrophy(e.g., co-A ligase deficiency), metachromatic leukodystrophy(arylsulfatase A deficiency) (e.g., symptomatic, or presymptomatic lateinfantile or juvenile forms), globoid cell leukodystrophy (Krabbe'sdisease; galactocerebrosidase deficiency), acid lipase deficiency(Wolman disease), cystic fibrosis, glycogen storage disease,hypothyroidism, sickle cell anemia, thalassemia (e.g., betathalassemia), Pearson syndrome, Pompe's disease, phenylketonuria (PKU),porphyrias, maple syrup urine disease, homocystinuria,mucoplysaccharidosis, chronic granulomatous disease and tyrosinemia andTay-Sachs disease or to treat solid tumors or other pathologicalconditions.

In other embodiments, the disease, disorder or condition is a disease,disorder or condition requiring replacement or repair of one or moretissues. For example, the combined stem cell populations of theinvention can be used in therapeutic transplantation protocols, e.g., toaugment or replace stem or progenitor cells of the liver, pancreas,kidney, lung, nervous system, muscular system, bone, bone marrow,thymus, spleen, mucosal tissue, gonads, or hair. The combined stem cellpopulations of the invention can also be used for augmentation, repairor replacement of, e.g., cartilage, tendon, or ligaments. For example,in certain embodiments, prostheses (e.g., hip prostheses) are coatedwith replacement cartilage tissue constructs grown from combined stemcell populations of the invention. In other embodiments, joints (e.g.,knee) are reconstructed with cartilage tissue constructs grown fromcombined stem cell populations. Cartilage tissue constructs can also beemployed in major reconstructive surgery for different types of joints(for protocols, see e.g., Resnick, D., and Niwayama, G., eds., 1988,DIAGNOSIS OF BONE AND JOINT DISORDERS, 2D ED., W. B. Saunders Co.). Thecombined stem cell populations of the invention can be used to repairdamage of tissues and organs resulting from trauma, metabolic disorders,or disease. In one embodiment, a patient can be administered a combinedstem cell population to regenerate or restore tissues or organs whichhave been damaged as a consequence of disease, e.g., to repair hearttissue following myocardial infarction.

In another embodiment, the combined stem cell populations of theinvention may be used to treat an individual who has received a lethalor sub-lethal dose of radiation. Such radiation may be accidentallyreceived, for example in a nuclear incident, whether work- oraggression-related, or therapeutic, for example, as part of a medicalprocedure. The particular type of radiation (e.g., alpha, beta, gamma)is not critical. The combined stem cell populations of the invention maybe used to ameliorate one or more symptoms of radiation sickness, forexample, nausea, loss of appetite, lethargy, dyspnea, decreased whiteblood cell count, chronic anemia, fatigue, weakness, paleness,difficulty breathing, feelings of malaise, and the like, whether suchsymptoms are indicative of recoverable or fatal radiation sickness. Inanother embodiment, the individual has one or more symptoms associatedwith acute radiation syndrome (ARS). The combined stem cell populationsof the invention may also be used to partially or fully reconstitute thehematopoietic system of an individual that has received a lethal orsub-lethal dose of radiation, such that the individual becomes partiallyor fully chimeric. Such chimerism may be temporary or permanent (e.g.,may persist for 1, 2, 3 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11months or longer). In a preferred embodiment, a combined stem cellpopulation of the invention is provided to the individual within thefirst 24 hours after exposure. The individual may be administered acombined stem cell population within the first hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, or 21hours after exposure to radiation. A combined stem cell population ofthe invention may also be administered within 2 days, 3 days, 4 days, 5days, 6 days, one week, 2 weeks, 3 weeks, 4 weeks or 5 weeks afterexposure to radiation.

The combined stem cell populations are expected to have ananti-inflammatory effect when administered to an individual experiencinginflammation. In a preferred embodiment, the combined stem cellpopulations of the invention may be used to treat any disease, conditionor disorder resulting from, or associated with, inflammation. Theinflammation may be present in any organ or tissue, for example, muscle;nervous system, including the brain, spinal cord and peripheral nervoussystem; vascular tissues, including cardiac tissue; pancreas; intestineor other organs of the digestive tract; lung; kidney; liver;reproductive organs; endothelial tissue, or endodermal tissue.

The combined stem cell populations may also be used to treat autoimmuneor immune system-related disorders, including those associated withinflammation. Thus, in certain embodiments, the invention provides amethod of treating an individual having an autoimmune disease orcondition, comprising administering to such individual a therapeuticallyeffective amount of the cells or supplemented cell populations of theinvention, wherein said disease or disorder can be, but is not limitedto, diabetes, amyotrophic lateral sclerosis, myasthenia gravis, diabeticneuropathy or lupus. In related embodiments, the combined stem cellpopulations of the invention may be used to treat immune-relateddisorders, such as chronic or acute allergies.

Combined stem cell populations may also be administered to a nominallyhealthy individual to increase the individual's overall health andwell-being.

Therapeutic or prophylactic treatment of an individual with combinedstem cell populations may be considered effective if the disease,disorder or condition is measurably improved in any way. Suchimprovement may be shown by a number of indicators. Measurableindicators include, for example, detectable changes in a physiologicalcondition or set of physiological conditions associated with aparticular disease, disorder or condition (including, but not limitedto, blood pressure, heart rate, respiratory rate, counts of variousblood cell types, levels in the blood of certain proteins,carbohydrates, lipids or cytokines or modulation expression of geneticmarkers associated with the disease, disorder or condition). Treatmentof an individual with the stem cells or supplemented cell populations ofthe invention would be considered effective if any one of suchindicators responds to such treatment by changing to a value that iswithin, or closer to, the normal value. The normal value may beestablished by normal ranges that are known in the art for variousindicators, or by comparison to such values in a control. Introductionof a combined stem cell population of the invention for the purposes ofengraftment, e.g., hematopoietic engraftment, would be consideredsuccessful if the individual to whom the combined stem cell populationis introduced exhibits any indications of engraftment (e.g., markers ofengrafted cells appearing in biopsy or tissue samples, or blood sample;detection of one or more biochemical functions performed by theengrafted cells, etc.). In medical science, the efficacy of a treatmentis also often characterized in terms of an individual's impressions andsubjective feeling of the individual's state of health. Improvementtherefore may also be characterized by subjective indicators, such asthe individual's subjective feeling of improvement, increasedwell-being, increased state of health, improved level of energy, or thelike, after administration of the stem cells or supplemented cellpopulations of the invention.

5.6 Stem Cell Bank

The methods described above, particularly the in vitro method (seeSection 5.1.1) may be performed on individual units of, for example,placental perfusate, placental stem cells, cord blood, cord blood stemcells, and the like, to produce combined stem cell populations for thetreatment of an individual in need of stem cells. As such, the assay maybe used as part of a method of stem cell banking or blood banking,including a cord blood banking, wherein providing stem cells is at leasta part of said banking. The assay may be performed on each of aplurality of units of placental stem cells, and stem cells from a secondsource, used or provided by a blood bank, stem cell registry, or similaroperation.

For example, in one embodiment, the invention provides a method of stemcell banking comprising providing a plurality of units of combined stemcell populations comprising a number of placental stem cells and stemcells from a second source, wherein said combined stem cell populationsexhibit improved or enhanced engraftment compared to a number of saidplacental stem cells or of said stem cells from a second source,equivalent to the number of cells in said combined stem cell population,alone. In a specific embodiment, said combined stem cell populations aregenerated by a method comprising providing a plurality of units ofplacental stem cells; providing a second plurality of stem cells from asecond source; matching each said units of placental stem cells with aunit of stem cells from a second source; and identifying a ratio of saidplacental stem cells to said stem cells from a second source in a totalnumber of cells that, when combined for a time and under conditionssufficient to allow the formation of colony-forming units, produces agreater number of colony-forming units than a number of said placentalstem cells or of said stem cells from a second source, equivalent tosaid total number of cells, alone. In a specific embodiment, said stemcells from a second source are cord blood or placental blood stem cells.In another specific embodiment, said stem cells from a second source areperipheral blood stem cells. In another specific embodiment, said stemcells from a second source are bone marrow stem cells. In anotherspecific embodiment, said placental stem cells and said stem cells froma second source are randomly matched. In another specific embodiment,said placental stem cells and said stem cells from a second source arematched based on a characteristic of said unit of placental stem cellsand of said unit of stem cells from a second source. In a more specificembodiment, said characteristic is the number of total nucleated cellsin said unit of placental stem cells and in said unit of stem cells froma second source. In another more specific embodiment, saidcharacteristic is the number of stem cells in said unit of placentalstem cells and in said unit of stem cells from a second source. Inanother more specific embodiment, said characteristic is animmunological marker displayed by said placental stem cells and by saidstem cells from a second source.

The invention further provides a bank of placenta-derived stem cells,e.g., a bank of units of placenta-derived stem cells and stem cells froma second source, wherein a number of said placenta-derived stem cellsand stem cells from a second source are provided together in a ratiothat produces more colony-forming units in a total number of cells,under conditions that allow the formation of colony-forming units, thana number of placental stem cells or said number of stem cells from asecond source, equivalent to said total number of cells, alone. In apreferred embodiment, the bank comprises a plurality of units ofplacenta-derived stem cells that are matched, or otherwise identified ascombinable with, one or more units of stem cells from a second source inratios, specific to the respective units, that, when the units arecombined, show greater numbers of colony-forming units in acolony-forming unit assay, or improved engraftment when transplantedinto a recipient, as compared to an equivalent number ofplacenta-derived stem cells or stem cells from a second source, alone.The bank can comprise separate, matched units of placenta-derived stemcells and stem cells from a second source, or units of combined stemcell populations.

Placenta-derived stem cells contained within such a bank, or withinunits of combined stem cell populations within such a bank, can be, forexample, cells contained within perfusate obtained directly from aplacenta, placenta-derived stem cells isolated from placental perfusateor enzymatic digestion of placenta and contained within a nucleated cellfraction, a population of placenta-derived stem cells isolated from theremainder of placenta cells according to, e.g., one or more cell surfacemarkers, or a population of stem cells cultured and/or expanded from anyof the foregoing. Stem cells from a second source can be containedwithin a tissue homogenate or other collection of tissue-specific cells,e.g., whole umbilical cord blood or placental blood, stem cells isolatedfrom the second source to any degree, or stem cells cultured and/orexpanded from any of the foregoing.

Preferably, the placental stem cells and stem cells from a second sourceare derived from the same individual. In a specific embodiment, saidstem cells from a second source are cord blood and/or placental bloodstem cells from the placenta from which the placental stem cells areobtained or derived. In a preferred embodiment, the bank comprises aplurality of units of combined stem cell populations comprisingplacental stem cells and stem cells from umbilical cord blood orplacental blood units, from the same individual, in ratios, specific tothe respective units, that produce greater numbers of colony-formingunits in a colony-forming unit assay, or improved engraftment whentransplanted into a recipient, for a total number of cells, compared toa number of placenta-derived stem cells or stem cells from a secondsource, equivalent to said total number of cells, alone.

Preferably, placenta-derived stem cells in the stem cell bank arecharacterized by at least one HLA marker. In a preferred embodiment, thebank comprises a plurality of units of HLA-characterizedplacenta-derived stem cells. In one embodiment, the invention provides astem cell bank comprising a plurality of units of placenta-derived stemcells, wherein said placenta-derived stem cells are identified by atleast one HLA marker. In a specific embodiment, said placenta-derivedstem cells are isolated from placental perfusate. In another specificembodiment, said placenta-derived stem cells are contained within apopulation of nucleated cells isolated from placental perfusate. Inanother specific embodiment, said placenta-derived stem cells are CD34⁺stem cells. In another specific embodiment, said placenta-derived stemcells are positive for CD105 or CD73, or bind antibodies SH2, SH3 and/orSH4. In another specific embodiment, said placenta-derived stem cellsare positive for OCT-4 and/or HLA-G.

In one embodiment, the stem cell bank of the invention comprises aplurality of units of blood or blood-derived stem cells, e.g., placentalblood or umbilical cord blood, or stem cells obtained from umbilicalcord or placental blood. Preferably, at least one, and preferably amajority, of the units of blood or blood-derived stem cells containedwithin the stem cell bank are, or can be, HLA-matched to at least one,or preferably a majority, of the units of placenta-derived stem cellscontained within the bank. Thus, in another specific embodiment, saidstem cell bank additionally comprises a plurality of units of blood orblood-derived stem cells. In another specific embodiment, at least oneunit of said plurality of units of blood or blood-derived stem cells isidentified by at least one HLA marker shared by one of said plurality ofunits of placenta-derived stem cells. In another specific embodiment, amajority of units within said plurality of units of placental blood orumbilical cord blood is identified by an HLA marker shared by a majorityof units within said plurality of units of placenta-derived stem cells.

The units of placenta-derived stem cells and units of blood-derived stemcells contained within the stem cell bank are preferably indexed andcross-matched for easy identification and combination to introduce intoa specific individual. For example, a specific individual having aparticular HLA marker, or HLA marker profile, can be matched to one ormore units of placenta-derived stem cells and, preferably, one or moreunits of blood-derived stem cells, e.g., umbilical cord blood orplacental blood. Preferably, the placenta-derived stem cells and bloodstem cells are combined to form a combined stem cell population of theinvention prior to administration to said individual. Such a combinedstem cell population may be produced according to the methods describedelsewhere herein.

The stem cell bank may comprise placenta-derived stem cells and/ormatched units of blood obtained from any number of individuals. Invarious embodiments, the stem cell bank of the invention may compriseunits of placental stem cells and/or units of blood, e.g., placentalblood and/or umbilical cord blood, obtained from at least 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000,400000, 500000, 600000, 700000, 700000, 800000, 900000 or 1000000, ormore, individuals.

5.7 Kits

The invention further provides kits that can be used to identify and/orprepare the combined stem cell populations of the invention. Such kitsenable the user to determine an appropriate ratio of placental stemcells and stem cells from a second source to use to prepare a combinedstem cell population. Such kits can be used to prepare combined stemcell populations that reflect the physiological status of the individualunit or units of placental stem cells, and stem cells from a secondsource, used to make the combined stem cell populations. In particular,such kits enable a user to perform a colony-forming unit assay usingplacental stem cells and stem cells from a second source.

Thus, in one embodiment, the invention provides a kit comprising, in asealed container, a population of placental stem cells and a pluralityof containers suitable for performing a colony-forming assay. In aspecific embodiment, said plurality of containers is a plurality ofwells in a tissue culture plate. Said plate may comprise at least 8, atleast 12, at least 24, at least 48, at least 96, or at least 128 wells.

In another specific embodiment, said kit comprises a set of instructionsfor the co-culture of placental stem cells and stem cells from a secondsource. In a more specific embodiment, said instructions compriseinstructions for culturing said placental stem cells and said stem cellsfrom a second source for the production of colony-forming units. Inanother more specific embodiment, said instructions compriseinstructions for co-culturing said placental stem cells and said stemcells from a second source in a plurality of ratios, and for selectingone of said plurality of ratios.

In another specific embodiment, said kit comprises one or morecontainers of medium suitable for the isolation of stem cells. Inanother specific embodiment, said kit contains one or more containers ofmedium suitable for the culture and/or differentiation of stem cellsinto colony-forming units. In a more specific embodiment, said medium isa methylcellulose-based or starch-based medium. In another more specificembodiment, said medium is a culture medium suitable for culturing stemcells. In an even more specific embodiment, said medium is Methocult GF⁺H4435 medium, RPMI 1640 medium supplemented with 2% fetal calf serum and1% Stemspan CC100 cytokine cocktail, Dulbecco's Modified Eagle's Medium(DMEM) or Iscove's Modified Dulbecco's Medium (IMDM).

In other specific embodiments, the kit comprises a scoring grid, whereinsaid scoring grid facilitates the counting of colony-forming units. Inanother more specific embodiment, said kit comprises a hemacytometer.

In another specific embodiment, the kit comprises a container suitablefor combining and storing placental stem cells and stem cells from asecond source in a ratio identified as described above. In more specificembodiments, said container is a blood bag.

In various other embodiments, the kit comprises one or more of adisposable (e.g., gloves, towelettes, and the like); a log for recordingresults; labels for containers, etc.

In another specific embodiment, said kit comprises statistical softwarefor determining which of a plurality of ratios of placental stem cellsto stem cells from a second source yields a significantly higher numberof colony-forming units than any other of said plurality of ratios.

6. EXAMPLES

6.1 Example 1

In Vitro Colony Forming Unit Assay

Total nucleated cells are isolated from a unit of cord blood byHetastarch separation. Total nucleated placental cells are obtained from750 milliliters of placental perfusate by Ficoll separation. The totalnucleated cells from placenta and cord blood are combined in triplicatein 35 mm culture dishes in Methocult GF⁺ H4435 medium (Stem CellTechnologies, Vancouver, Canada), or RPMI 1640 medium supplemented with2% fetal calf serum and 1% Stemspan CC100 cytokine cocktail (Stem CellTechnologies, Vancouver, Canada). Cells are combined in at least tworatios (e.g., 2×10⁵:2×10⁵; 1×10⁵:3×10⁵; 3×10⁵:1×10⁵), and are culturedfor 14 days. The morphology of the cells is then examined under phasecontrast microscope, and the total number of colony-forming units (e.g.,CFU-GM, CFU-L, CFU-M, CFU-G, CFU-DC, CFU-GEMM, CFU-E) are recorded. Adetermination is then made as to which ratio produces the highest numberof colony-forming units.

6.2 Example 2

Co-Culture Assay Using Hematopoietic Stem Cells

Ten HLA/donor matched placental perfusate and cord blood units werethawed and total nucleated cells (TNC) were counted on a Cell-Dyn 1700(Abbott Laboratories, Abbott Park, Ill.). The CFU assays of theco-culture experiments were studied in triplicate in 35 mm dishes inMethoCult GF+ H4435 Medium (StemCell Technologies, Vancouver, Canada).Mononuclear cells were seeded as follows: placental perfusate-derivedstem cells (PP) alone at 50 (low seeding group), 250 (medium seedinggroup) and 5000 (high seeding group)×10³/mL/dish; cord blood-derivedstem cells (CB) alone at 50×10³/mL/dish; and placental perfusate-derivedstem cells and cord blood-derived stem cells in co-culture, with50×10³/mL/dish cord-blood-derived cells in combination with 50, 250 and5000×10³/mL/dish placental perfusate-derived stem cells. Colony formingunit assays were read on day 14 after seeding. The increase in thenumber of total colony-forming units was calculated based on theformula: % increase in total colony-formingunits=CB/PP−(CB+PP)/(CB/PP)×100.

A total of 10 matched CB and PP samples were co-cultured. In 4 of the 10co-cultures, total CFU activity increased in counts per dish as comparedto CB or PP culture alone. The percentage increase of totalcolony-forming unit activity varied from 7.1% to 43.1% in the lowseeding group, 14.9% to 42.1% in the medium seeding group, and 24.4% to43.8% in the high seeding group.

6.3 Example 3

Pre-Clinical Studies to Evaluate the Hematopoietic ReconstitutionActivity of Human Placenta Perfusate and Umbilical Cord Blood byAccessing SCID-Repopulating Cell Activity

6.3.1 Introduction

The stem cell properties of cells present in the UCB and HPSC wereevaluated using quantitative assessments in a xenogeneic transplantmodel using immunodeficient NOD/SCID mice. Reported herein are resultsof the initial experiments to determine the frequency and absolutenumber of NOD/SCID repopulating cells in UCB and HPSC by limitingdilution transplant studies.

6.3.2 Materials and Methods

6.3.2.1 Collection and Cryopreservation of UCB Units

Briefly, after informed consent of the mother was obtained, the UCB wereharvested at hospital in a triple-bag system containingcitrate/phosphate/dextrose solution. The units were stored and processedat room temperature within 48 hours of blood collection. AHetastarch-based method was used to perform volume reduction and RBCdepletion. The final TNC were frozen in a cryobag containing 40 mL of10% DMSO and autologous plasma in LN2 tank in vapor phase.

6.3.2.2 Placental Perfusion and Cryopreservation of HPSC Units

Placental stem cells were collected by placental perfusion according tothe methods disclosed in United States Application Publication Nos.2002/0123141 and 2003/0032179, each of which is incorporated herein byreference in its entirety. Briefly, placentas from the umbilical cordblood donors were drained of umbilical cord and perfused with 0.9% NaClsolution at controlled pressure. A total of 750 mL perfusate wasobtained. The cells were concentrated and separated by gradientseparation (Ficoll-Hypaque) to deplete RBC and cell debris.

6.3.2.3 Cell Counting and Viability

Cell counts were performed with automated cell analyzers (Cell-Dyn 1700or Cell-Dyn3200, Abbott; Wiesbaden, Germany) and by manual counting. Theviability of the cells was determined using trypan blue exclusion.

6.3.2.4 Phenotyping of Umbilical Cord Blood (UCB) and PlacentalPerfusate (HPSC) Units

Single donor matching units of UCB and HPSC were maintained in liquidnitrogen until the time of use. On the day of transplant, the units werethawed. The viability of freshly thawed UCB and HPSC units wasdetermined using trypan blue. Post-thaw recovery of total nucleated cellcounts was also performed. Prior to transplant, an aliquot (0.5 ml) offreshly thawed cells was then used for FACS analysis for the followingcell surface markers: CD34, CD38, CD33, CD14, CD7, CD3, CD56, CD10 andCD19.

6.3.2.5 Limiting Dilution NOD/SCID Repopulating Cell Assay

Quantitative studies using limiting dilution SCID repopulating cell(SRC) assays were carried out using NOD/SCID mice at 8-10 weeks of age.The mice were irradiated at 325-350 cGy with irradiation from a linearaccelerator at an exposure rate of 20 cGy/min prior to transplantation.Mice were then transplanted intravenously via the lateral tail vein with200 μl of cells from cord blood or placental perfusate or thecombination of cord blood and placental perfusate. Transplants comprisedapproximately 2-10×10⁵ stem cells per kg (non-expanded) or 1-2×10⁶ stemcells per kg (expanded). Four cell doses were used in order to calculatethe frequency of repopulating cells and 6 mice per group weretransplanted. Mice were then analyzed for human cell engraftment atthree weeks post transplant and at 10 weeks post transplant. Cells wereobtained from 25 μl of aspirated bone marrow harvested from the femurand then analyzed by FACS analysis for human lympho-myeloid engraftment.For each aspiration a tuberculin syringe with a 28-gauge needle wasprepared containing approximately 30-40 uL of PBS. At 10 weeks, micewere sacrificed and cells harvested from both femurs, both tibiae, andthe thymus for engraftment analysis. Additionally, in the secondexperiment carried out, necropsy was performed for all mice in thehighest cell dose group and tissues collected for histology and presenceof human cells. The tissues collected included: spleen, liver, lung,brain, heart, skeletal muscle, kidney and thymus. Engraftment wasdefined as ≧0.5% CD45+ cells.

6.3.3 Results

6.3.3.1 TNC, Viability and TNC Recovery of HPSC and UCB Units Used inExperiments

Two matching units of HPSC and their matching UCB units were usedindependently for each experiment. Table 1 shows the TNC and post-thawviability and TNC recovery rate of these cells. The two UCB units haveTNC of 1237×10⁶ and 778×10⁶ and viability of 82% and 83%, respectively.HPSC units have a relatively lower TNC counts (752.5×10⁶ and 661.5×10⁶)and viability (67% and 66% respectively) compared to the UCB units.

TABLE 1 TNC and viability of two matching placenta and UCB units used inNOD/SCID BTM engraftment experiments Exp-1 UCB Exp-1 PP Exp-2 UCB Exp-2PP Prefreeze TNC (×10⁶) 1237 752.5 778.0 661.5 Post-thaw Viability 83%67%  82%  66% Post-thaw TNC Recovery 99% 53% >100% >100%

6.3.3.2 Phenotypic Analysis of UCB and HPSC

Table 2 outlines the results of the phenotypic analysis of the cordblood and placental perfusate cells prior to transplant. As expected,there was variability between the cord blood donors in experiment 1 and2 with respect to the percent of CD34+ cells, with 0.56% of the cellsbeing CD34+ in experiment 1 versus 1.67% CD34+ cells in experiment 2.These differences reflect the natural variability in TNC and numbers ofCD34⁺ cells in umbilical cord blood between donors. In either case, thepercent of CD34+ cells was greater in the cord blood than the placentalperfusate. The cord blood was lower in the myeloid markers (CD33 andCD14), but higher in lymphoid markers (CD3 and CD7). A significantlyhigher number of cells in HPSC express CD10 than in cord blood.

TABLE 2 FACS analysis of matching UCB and HPSC units used in theNOD/SCID mice BTM engraftment experiments Exp-1 UCB Exp-1 PP Exp-2 UCBExp-2 PP CD34+ 0.56% 0.28%  1.67%  0.46% CD34+CD38+ 0.56% 0.28%  1.67% 0.46% CD33+ 26.0%   60% 28.00% 76.00% CD14+ 17.0%   46% 22.40% 58.40%CD7+ 38.5% 10.5% 63.00% 18.00% CD3+ 35.2% 11.8% 72.00% 29.00% CD56+ 7.7%  3.7% 16.50% 12.00% CD10+ 16.8% 53.0%  9.50% 59.00% CD19+ 15.6%11.0%  8.80% 13.00%

6.3.3.3 Engraftment of Human Cells in NOD/SCID Mice

Table 3 shows the cell doses of TNC infused to NOD/SCID mice in twoindependent experiments (Experiment 1 and 2). In both cases, equivalentnumbers of CD34+ cells from UCB or HPSC were used in all mice receivedthe UCB or HPSC. The TNC cell doses required for that number of CD34+cells were infused to the mice accordingly. Six mice were used perdosage group.

TABLE 3 Cell dose of TNC transplantation in NOD/SCID mice CD34TNC/mouse: equivalent UCB PP UCB + PP Mice/grp A. Experiment 1 1.5 × 10⁵15 × 10⁶   15 × 10⁶  30 × 10⁶ 6   3 × 10⁴ 3 × 10⁶   3 × 10⁶   6 × 10⁶ 6  6 × 10³ 6 × 10⁵   6 × 10⁵ 1.2 × 10⁶ 6 1.2 × 10³ 1.2 × 10⁵   1.2 × 10⁵2.4 × 10⁵ 6 B. Experiment 2 2.4 × 10⁵ 24 × 10⁶   24 × 10⁶  48 × 10⁶ 6  8 × 10⁴ 8 × 10⁶   8 × 10⁶  16 × 10⁶ 6 1.1 × 10⁴ 1.1 × 10⁶   1.1 × 10⁶2.4 × 10⁶ 6 8.9 × 10³ 8.9 × 10⁵   8.9 × 10⁵ 1.8 × 10⁶ 6

FIG. 1 shows the summary of FACS analysis of engrafted human cells inmice bone marrow using CD45 antibodies in two independent experiments.At week 3 and week 10, mice bone marrow aspirates were analyzed for thepresence of human CD45⁺ cells by FACS. Very low or undetectable numbersof human CD45⁺ cells were found in mice receiving placental cells alonein both time points in any cell doses. In contrast, at both time points,human cell engraftment was seen in mice transplanted with cord bloodalone and with the combination of cord blood and placental perfusate. At3 weeks, there was no significant difference seen in the level of humanengraftment between the cord blood and combined cord blood and placentalperfusate. However, at 10 weeks, the degree of human cell engraftmentwas significantly enhanced in mice receiving both cord blood cells andplacental perfusate (p=0.3 in experiment 1 and p=0.0002 in experiment2), as compared to engraftment in mice receiving umbilical cord bloodstem cells or placental stem cells alone, indicating that placental stemcells enhance engraftment of the stem cells from a second source, e.g.,umbilical cord blood.

To determine if the human engraftment cell included lymphomyeloidlineages, FACS analysis was also used to analyze co-expression of CD19,CD33 and CD7 in CD45⁺ cells from mouse bone marrow. The results fromthis experiment are shown in FIG. 2. These results show that the marrowof mice receiving both UCB and UCB+HPSC contained engrafted lymphoid andmyeloid cells.

6.3.3.4 SCID Repopulating Cell (SRC) Frequency

The SCID Repopulating Cell frequency is the ratio of primitivehematopoietic stem cells, able to engraft and repopulate thehematopoietic system of an individual, to the total number of cellstransplanted. The ratio provides an indication of the relative abilityof a cell population to provide engraftable cells to, for example, anirradiated individual. Table 4 lists the SRC calculations fromexperiment 2 (see above). These numbers were calculated by limitingdilution transplants and application of the L-Calc software fromStemCell Technologies. These studies did not demonstrate an enhancementof the SRC frequency, but as noted above, did show significantenhancement of overall human engraftment upon co-infusion of cord bloodand placental perfusate. Thus, the data, in this instance, indicate thatco-infusion of the placental perfusate with the UCB enhances stem cellengraftment, rather than increasing the overall number of stem cells.

TABLE 4 Estimation of SRC frequency from UCB and HPSC Frequency RangeWK-3 WK-3 UCB 1/17,791,258 12,060,000 to 26,245,000 PP NA NA UCB + PP1/28,728,138 19,782,000 to 41,719,000 WK-10 WK-10 UCB 1/2,859,018 1,867,000 to 4,376,000 PP NA NA UCB + PP 1/7,864,065   5,186,000 to11,923,000

6.3.3.5 Engraftment of Human Cells in Non-Bone Marrow Tissues

To determine if human cells from UCB, HPSC or UCB+HPSC are engrafted inmouse tissues other than the bone marrow, the presence of human cells inexperimental mouse thymus was determined by FACS analysis, andimmunohistochemical staining was performed on mouse spleen tissue.

In experiment 1, FACS analysis of cells from mouse thymus showed thatone mouse out of six co-infused with UCB and HPSC showed 0.8% humanCD45⁺ cells. In experiment 2, one mouse out of six infused with UCB(dose 2) showed 8% of CD45⁺ cells, but no CD3⁺ or CD7⁺ cells. However,in the UCB+HPSC group, all six mice showed human engraftment with 3-23%CD45+ cells and one of these mice has shown CD3/CD7 positive cells.

Thin sections of the mice spleen were examined to detect the presence ofhuman cells by staining with anti-vimentin and anti-CD45 antibodies thatrecognize human but not mouse proteins. Smooth muscle actin antibodiesthat recognize both human and mouse proteins were used as a positivecontrol and IgG1 and IgG2a isotypes were used as negative controls. Theresults of the staining from each engraftment group are shown in Table5.

TABLE 5 Detection of human cells in the spleen of NOD/SCID mice byimmunohistochemstry Smooth Mouse muscle Number Product Vimentin CD45actin IgG1 IgG2a 304 UCB-1 & 3+ 2+ 2+ — — PP-1 305 UCB-1 & 2+ 1+ (few)2+ — — PP-1 306 UCB-1 & 3+ 2+ 2+ — — PP-1 307 UCB-1 & 3+ 2+ 2+ — — PP-1308 UCB-1 & 3+ 2+ 2+ — — PP-1 350 PP-1 — — 2+ — — 351 PP-1 — — 2+ — —352 PP-1 — — 2+ — — 353 PP-1 — — 2+ — — 354 PP-1 ± (very — 2+ — — few)355 PP-1 — — 2+ — — 370 UCB-1 2+ — 2+ — — 371 UCB-1 1+ (few) — 2+ — —372 UCB-1 2+ 1+ (few) 2+ — — 373 UCB-1 ± (very — 2+ — — few) 374 UCB-11+ (few) — 2+ — — 375 UCB-1 1+ (few) — 2+ — — Human NA 3+ 3+ 2+ — —tonsil

Cells in the mouse spleen expressing human vimentin, a mesenchymal cellmarker, were detectable in all mice receiving UCB cells alone. Vimentinstaining was barely detectable in the mice receiving HPSC alone.However, significantly higher levels of vimentin staining were detectedin the mice receiving both UCB and HPSC cells. Similar results werefound when the spleen tissue was stained with antibodies to CD45, ahematopoietic cell marker. Smooth muscle actin (positive control)staining of the mouse spleen showed a uniform level of staining on alltissues. The isotype negative control antibodies did not stain thetissues.

6.3.4 Discussion

In these experiments, co-infusion of placental cells with cord bloodcells from the same donor was shown to enhance the level of human stemcell engraftment in mice at 10 weeks over infusion of cord blood orplacental cells alone. The enhanced human cell engraftment in NOD/SCIDmice was also found in tissues including thymus and spleen. Theengrafted cells are shown to include both myeloid and lymphoid cells.Engrafted human cells stained positive with vimentin in mouse spleens,indicating that the engraftment of human stem cells is enhanced by theUCB-HPSC co-infusion.

6.4 Example 4

Engraftment in NOD/SCID Mice

A dose range pilot study was performed in which combinations of humanumbilical cord blood cells and placental cells were administered tosub-lethally-irradiated NOD/SCID mice in different ratios, and in whichthe degree of engraftment of, and repopulation by, human cells wasdetermined.

Six groups of NOD/SCID mice, a model of human transplant engraftment,were sublethally irradiated at 400 cGy and dosed intravenously with oneof three doses of cord blood cells and placental cells, based on thenumber of live total nucleated cells, at either a 3:1 or 1:1 ratio ofcord blood cells to placental cells. FACS analysis was performed on thecells following combination and injection. Mice were monitored forengraftment of human cells by blood and bone marrow sampling at 10 weeksafter administration.

Mice were administered one of the combinations of cord blood calls andplacental cells shown in Table 6:

TABLE 6 Combinations of umbilical cord cells and placental cellsadministered to NOD/SCID mice Quantitative SCR Assay in NOD/SCID MiceWith Cord Blood Cells and Placental Cells* Cord Blood Placental Numer ofCells (Live Cells Total Cells Dose Volume Mice Group Cell Ratio TNC)(Live TNC) (Live TNC) (μL) (Males) Subset A: 1 1:1 4.5 × 10⁶ 4.5 × 106 9 × 10⁶ 200 10 2 1:1   9 × 10⁶   9 × 106 18 × 10⁶ 200 10 3 1:1  18 ×10⁶  18 × 106 36 × 10⁶ 200 10 Subset B: 1 3:1 4.5 × 10⁶ 1.5 × 106  6 ×10⁶ 200 10 2 3:1   9 × 10⁶   3 × 106 12 × 10⁶ 200 10 3 3:1  18 × 10⁶   6× 106 24 × 10⁶ 200 10 *Cell ratio subsets contained single units at eachdose level (i.e., groups 1 and 4 used the same unit, groups 2 and 5 usedthe same unit, and groups 3 and 6 used the same unit). At the highestdoes, pooling was required.Materials and Methods

Animals were handled in accordance with DHHS Publication No. (NIH) 86-23(Revised, 1985) and the U.S. Department of Agriculture through theAnimal Welfare Act (7 U.S.C. 2131), 1985 and Animal Welfare Standardsincorporated in 9 C.F.R. Part 3, 1991.

NOD/SCID male mice (Taconic Laboratories, Germantown, N.Y.), all between7 and 10 weeks old, were sub-lethally irradiated at 400 cGy using a¹³⁷Cesium source at a rate of about 171 cGy/min. Unanesthetized animalswere placed into a Mark I Model 68A Cesium irradiator for the 2.34minute irradiation interval.

Human placental cells and umbilical cord blood cells were isolated bypositive pressure collection (PPC) or negative pressure collection(NPC), and were cryopreserved prior to administration. Cells were thawedin a 37° C. water bath and diluted, then stored on wet ice. The diluentfor the cells comprised 5% dextran (Baxter) and 2.5% human serum albumin(Bayer). Cells were counted and assayed for viability. Cells wereadministered in a single dose through the tail vein of each mouse. Themice were housed under standard conditions and sacrificed at 10 weekspost-irradiation to analyze engraftment.

FACS analysis of marrow and thymus was performed for evidence of humancell engraftment, by assessment for frequency of CD45⁺ cells, as well asfrequency of CD34⁺, CD38⁺, CD19⁺, CD33⁺, CD7⁺ and CD3⁺ cells. Cells werecounted and about 500,000 cells were stained per well, at two wells persample. Mouse Fc block (purified mouse IgG) was added at 1 μg permillion cells, to reduce non-specific binding. Antibodies were added atabout 1 μg per million cells. One well contained antibodies for CD45,CD34, CD38 and CD19, and the second contained antibodies for CD45, CD33,CD7 and CD3. Isotype controls for each antibody were also used, at about1 μg per million cells. Following antibody staining, the cells wereincubated for 30 minutes at 2-8° C., washed three times with phosphatebuffered saline, 1% bovine serum albumin and 0.05% sodium azide, fixedwith 1% paraformaldehyde, and stored in the dark at 2-8° C. untilanalysis. Samples were analyzed by a Becton-Dickinson FACSCalibur withforward- and side-scatter gates set to exclude debris and clumps.Optimal voltage settings and compensations were determined by isotypecontrols.

Vimentin immunostaining was performed on paraffin sections of mousesternum using a human-specific vimentin antibody. Scoring was performedsemi-quantitatively using the following scale:

-   -   Score=0: No positive cells    -   Score=0.5: One or two positive cells, likely positive but cannot        be ruled out as    -   Score=1: 2-20 scattered positive cells    -   Score=2: Approximately 20-100 scattered or clustered positive        cells throughout the tissue    -   Score=3: More than 100 positive cells, but making up less that        50% of tissue    -   Score=4: More than 50% of marrow cells are positive        Results:

Repopulation data are summarized in Table 7, and FACS data is summarizedin Table 8. Repopulation was evident to some degree in all animalgroups, and the effect appeared to be dose-dependent. The meanpercentage of cells positive for each marker was compared at the twodifferent ratios. CD7, CD33, and CD34 showed statistically significantdifferences between the two ratios, with the 1:1 ratio showing a lowerpercentage of positive cells than the 3:1 ratio

Vimentin staining. Almost all of the sternum sections were composed of5-6 marrow cavities roughly rectangular in shape, showing some variationin size and shape and surrounded by bony and cartilage tissue. Allvimentin positive cells were seen within the bone marrow along withouter erythroid and myeloid precursors in various stages or maturation.No vimentin positive cells were observed in the negative control. Eachmarrow cavity was scored individually. Generally, the vimentin scorecorrelated well with the dose of cells injected. Both Groups 3 and 6,having the highest number of stem cells, had similar high scores of 3.4.At low and medium dose levels, there was a slight difference between thegroups injected with the same number of cells. For example, the meanscore for Group 4 (3:1 ratio cord blood cells to placental cells) wasslightly higher than Group 1 (1:1 ratio), and the mean score for Group 5(3:1 ratio) was slightly higher than group 2 (1:1).

Conclusions

Flow cytometry and immunohistochemical evaluations of bone marrowdemonstrated substantial repopulation in a cell dose-dependent manner.Differences between the two cell ratios rose to the level of statisticalsignificance for CD7, CD33 and CD34 engraftment. Where significantdifferences existed, animal receiving 3:1 cord blood to placental cellratio had a higher degree of repopulation than animals treated with a1:1 ratio.

TABLE 7 Bone Marrow Repopulation Group Tibia Femur 1  9/10  6/10 2 6/75/7 3 9/9 9/9 4  4/10  5/10 5 7/7 7/7 6 9/9 9/9

Numerator indicates the number of animals per group in which thepercentage of CD45+ cells was greater than or equal to 0.5%. Denominatorindicates the number of animals per group in which flow cytometry wasperformed.

TABLE 8 Summary of FACS analysis results. SUMMARY OF FACS ANALYSIS BYTREATMENT GROUP CD34 CD45 CD19 CD38 CD33 CD3 CD7 Group T F A T F A T F AT F A T F A T F A T F A 1 0.0 0.0 0.0 2.2 0.8 1.5 0.1 0.1 0.1 0.0 0.00.0 0.1 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 2 1.1 0.6 0.9 8.0 7.3 7.7 6.65.8 6.2 7.8 6.6 7.2 0.6 0.6 0.6 0.0 0.0 0.0 0.0 0.0 0.0 3 4.9 7.4 6.235.1 34.8 35.0 30.6 34.4 32.5 37.5 42.3 39.9 5.2 7.1 6.2 0.0 0.0 0.0 0.00.2 0.1 4 1.2 0.9 1.1 4.8 1.5 3.2 3.9 0.3 2.1 5.5 1.2 3.4 2.4 1.7 2.10.0 0.0 0.0 0.3 0.4 0.4 5 2.6 2.8 2.7 13.1 10.1 11.6 10.4 5.2 7.8 15.610.6 13.1 2.0 2.7 2.4 0.0 0.0 0.0 0.4 0.4 0.4 6 10.2 10.7 10.5 47.9 46.647.3 35.9 35.6 35.8 51.0 51.8 51.4 11.0 10.4 10.7 0.0 0.0 0.0 1.2 1.11.2 T—Tibia; F = Femur; A = Average of Tibia and Femur. Numbers indicatethe percentage of cells staining positive for the indicated human cellsurface marker

6.5 Example 5

Hematopoietic Reconstitution in NOD/SCID Mice

Colony formation assays performed with CD34⁺ placenta-derived stem cells(HPDSC) have demonstrated the presence of functional hematopoietic stemand progenitor cells in placental perfusate. In addition, there is datasuggesting that HPDSC contains other novel stem cell populations withmore immature characteristics compared to umbilical cord blood (UCB).

An experiment was carried out to determine the engraftment potential ofplacental perfusate stem cells in a xenogenic transplant model usingimmunodeficient NOD/SCID mice. The first part of the study evaluated theengraftment potential of placental perfusate cells and umbilical cordblood cells alone or in combination, with control groups receivingpurified CD34⁺ cells. The second part of the study evaluated whether theinfluence of placental perfusate stem cells on enhanced engraftment isdue to increased numbers of repopulating cells or the presence offacilitator cells. In this experiment, three groups of mice receivedeither UCB alone, HPDSC alone, or a combination of HPDSC and UCB. Micereceived the same number of CD34⁺ cells in each group. Separate groupsof mice also received a combination of HPDSC and UCB, in which eitherthe HPDSC or UCB cells had been irradiated to prevent repopulatingability, but to preserve any facilitator effect. Because there is knownvariability in the SCID repopulating ability between individual units ofcord blood and placental perfusate, multiple pooled units were used inthese experiments.

Methods and Experimental Design

Male NOD/SCID mice 8-10 weeks old were obtained from Jackson Laboratory.Mice were handled aseptically and housed in micro isolator cages inaccordance with standard laboratory practice. Mice were offered waterand food ad libitum.

Frozen bags of HPDSC and frozen bags of human umbilical cord blood (UCB)were supplied by Celgene Cellular Therapeutics.

On the day of transplantation, HPDSC and UCB units were removed fromliquid nitrogen and thawed. After washing, total nucleated cells number(TNC) and viability was determined for each unit. In the secondrepopulation study part, HPDSC and UCB cells were thawed and prepared ina manner similar to that of the first part, except that HPDSC or UCBcells were irradiated in some of the dose groups. Combinations of HPDSCand UCB cell preparations were prepared by mixing appropriate amounts ofHPDSC and UCB cells.

Cell counts were performed with automated cell analyzers (Cell-Dyne 1700or Cell Dyne 320, Abbot; Wiesbaden, Germany). The viability of cellpreparation was determined by Trypan Blue exclusion method.

SCID repopulating cell assays were carried out in NOD/SCID mice at 8-10weeks of age. The mice were irradiated at 325-350 cGy with irradiationfrom linear accelerator at an exposure rate of 20 cGy/min prior totransplantation. Mice were then transplanted intravenously via lateraltail vain with 200 μL of UCB or human placental perfusate cells orcombination of UCB and human placental perfusate cells. Engraftmentanalysis was performed at 4 weeks and 12 weeks after transplant.

Analysis of engraftment of human cells was performed by flow cytometricanalysis. In brief, samples were stained with an antibody, fixed with 1%paraformaldehyde, and analyzed by using Becton Dickson FACSCalibur forhuman CD45 and panel of other lineage cell surface markers, includingCD34, CD7, CD33, CD10, CD7 and CD3. Optimal voltage settings andcompensations were determined by isotype control. Four weeks engraftmentanalysis was performed with bone marrow aspirate obtained fromanesthetized mice and at 12 weeks animals were sacrificed and bonemarrow cells were flushed from femurs and tibias. Mice were consideredengrafted if the percentage of human CD45 was >0.5%.

Experimental Design. On the day of transplantation animals wereirradiated and randomized into different treatment groups, as shown inTables 9 and 10:

TABLE 9 Repopulation study Part (A) CD34#/mouse Group Mice/Group Dosevolume ACTUAL UCB 13 200   1 × 10⁵ HPDSC 13 200 5.1 × 10⁴ UCB + HPDSC 13200 1 × 10⁵ + 5.1 × 10⁴ Control Hi 6 200 2.5 × 10⁵ Control Lo 5 200 1.25× 10⁵ 

TABLE 10 Repopulation study Part (B) Mice/ CD34#/mouse - CD34#/mouseTNC/mouse - Group Group Estimated ACTUAL ACTUAL(10⁶) UCB 15 3.0 × 10⁵CD34/mouse 2.7 × 10⁵ CD34/mouse 28.4 PP1 15 3.0 × 10⁵ CD34/mouse 2.6 ×10⁵ CD34/mouse 17.72 UCB + PP1 15 3.0 × 10⁵ CD34/mouse 2.7 × 10⁵CD34/mouse 14.8 + 8.86 (same total CD34 cell dose) (1.5 + 1.5) (1.4 +1.3) UCB^(irr) + PP1 15 3.0 × 10⁵ CD34/mouse 2.7 × 10⁵ CD34/mouse 14.8 +8.86 (same total CD34 cell dose) (1.5 + 1.5) (1.4 + 1.3) UCB + PP1^(irr)15 3.0 × 10⁵ CD34/mouse 2.7 × 10⁵ CD34/mouse 14.8 + 8.86 (same totalCD34 cell dose) (1.5 + 1.5) (1.4 + 1.3) Control 5 3.0 × 10⁵ CD34/mouseIrr = irradiated cellsResults

Post thaw cell viability. Post thaw cell viability of UCB and HPDSC wasmore than 70% for the units used in repopulation study.

Human Cell Engraftment in NOD/SCID Mice. Human cell engraftment (>0.5%CD45) was observed in all groups 4 weeks post infusion, including HPDSCalone, with 2 out of 6 mice positive for engraftment in the UCB group(mean CD45% of 0.62%), 2 out of 8 mice in the HPDSC group (mean CD45% of0.52%), and 8 out of 9 mice in the group that received both UCB andHPDSC (mean CD45% of 2.84%). There was a significant increase in humanengraftment observed when comparing either the HPDSC group alone to theUCB+HPDSC group (p=0.006) and the UCB group to the UCB+HPDSC group(p=0.02). At 12 weeks post transplant, sustained engraftment in theHPDSC group alone was not observed with only 1 out of 8 animalsengrafted at >0.5% CD45. In contrast, although there was no statisticaldifference observed in the overall level of human engraftment betweenmice that received UCB alone versus the UCB+HPDSC group (mean CD45% of15.1% and 13.1%, respectively; p=0.82), only 3 out of 6 mice wereengrafted in the UCB group as compared to 9 out of 9 mice in theUCB+HPDSC group. Mice engrafted with human cells also showed engraftmentof lymphomyeloyid and other lineage cell types (Tables 11 and 12). Thesedata indicate that co-infusion of HPDSC and UCB results in significantenhancement of both short- and longer-term human engraftment as comparedto HPDSC or UCB alone.

TABLE 11 Percent engraftment of lymphomyeloid and other lineage markerscells in bone marrow of NOD/SCID mice after 4 weeks of intravenoustransplantation of human placenta derived stem cells and umbilical cordblood alone or in combination Percent human cell Control Engraftment UCBHPDSC UCB + HPDSC (Hi) Control(Low) CD45 0.62 ± 0.92 0.52 ± 0.76 2.84 ±1.89 1.33 ± 0.90 0.09 ± 0.14 CD33 0.51 ± 0.78 0.43 ± 0.67 2.41 ± 1.520.91 ± 0.68 0.02 ± .05  CD19 0.17 ± 0.24 0.15 ± 0.31 0.76 ± 1.0  0.44 ±0.43 0.04 ± 0.08

TABLE 12 Percent engraftment of lymphomyeloid and other lineage markerscells in bone marrow of NOD/SCID mice after 12 weeks of intravenoustransplantation of human placenta derived stem cells and umbilical cordblood alone or in combination. Percent human cell Control EngraftmentUCB HPDSC UCB + HPDSC (Hi) Control(Low) CD45  15.0 ± 22.09 0.65 ± 1.5113.09 ± 11.56 11.3 ± 8.11 0.04 ± 0.04 CD34 3.67 ± 5.55 0.21 ± 0.54 3.21± 3.41 2.76 ± 2.08 0.01 ± 0.01 CD33 6.33 ± 9.18 0.40 ± 1.04 5.61 ± 5.194.18 ± 3.56 0.01 ± 0.01 CD19  9.96 ± 16.66 0.30 ± 0.70 8.52 ± 9.97 8.02± 5.67 0.02 ± 0.03 CD10 12.02 ± 17.51 0.26 ± 0.59  8.74 ± 10.04 8.28 ±5.60 0.02 ± 0.02 CD7 1.05 ± 1.65 0.21 ± 0.32 1.01 ± 1.36 1.05 ± 0.780.01 ± 0.01 CD3 0.14 ± 0.15 0.03 ± 0.03 0.10 ± 0.07 0.10 ± 0.06 0.02 ±0.01

Facilitator effect. Enhanced human engraftment was seen in the UCB+HPDSCgroup as compared to the UCB or HPDSC group alone (Tables 13 and 14).Furthermore, although the group of mice that received irradiated HPDSCwith UCB received half the number of functional CD34 cells per mice thanthe group of mice that received CB alone or CB+PP1, there was equivalenthuman engraftment in this group, suggesting a facilitator function ofthe HPDSC.

Delayed engraftment following cord blood transplantation remains asignificant clinical problem, even in the case of double unitmyeloablative cord blood transplantation, where the median time toneutrophil engraftment is about 23 days. These results also suggestclinical investigation of co-infusion of HPDSC with either single ordouble cord blood units for transplantation as a potential method tofacilitate more rapid engraftment.

TABLE 13 Percent engraftment of lymphomyeloid and other lineage markerscells in bone marrow of NOD/SCID mice after 4 weeks of intravenoustransplantation of human placenta derived stem cells and umbilical cordblood alone or in combination Percent human cell UCB + HPDSC EngraftmentUCB HPDSC UCB + HPDSC UCBirr + HPDSC irr Control CD45  11.0 ± 11.52 0.69± 0.70 6.84 ± 6.61 0.31 ± 0.48 16.48 ± 19.62 15.83 ± 11.25 CD34 5.45 ±5.50 0.34 ± 0.34 3.72 ± 3.82 0.08 ± 0.13  9.43 ± 13.99 6.90 ± 5.32 CD335.94 ± 5.59 0.52 ± 0.59 5.17 ± 5.26 0.26 ± 0.44 11.79 ± 16.62 6.41 ±5.10 CD19 5.65 ± 8.07 0.09 ± 0.10 2.06 ± 1.92 0.06 ± 0.10 5.75 ± 5.868.23 ± 5.52

TABLE 14 Percent engraftment of lymphomyeloid and other lineage markerscells in bone marrow of NOD/SCID mice after 12 weeks of intravenoustransplantation of human placenta derived stem cells and umbilical cordblood alone or in combination Percent human cell UCB + HPDSC EngraftmentUCB HPDSC UCB + HPDSC UCBirr + HPDSC irr Control CD45 41.86 ± 28.7015.10 ± 23.75 48.29 ± 28.18 0.68 ± 0.66 51.62 ± 29.91 33.37 ± 19.18 CD348.78 ± 6.20 2.48 ± 3.35 9.32 ± 7.70 0.08 ± 0.09 8.20 ± 6.38 10.29 ±5.77  CD33 7.98 ± 5.12 2.66 ± 4.01 8.32 ± 6.29 0.17 ± 0.20 6.16 ± 4.354.23 ± 2.72 CD19 36.99 ± 25.69 13.48 ± 21.35 41.25 ± 0.49  0.49 ± 0.6947.04 ± 27.07 28.31 ± 16.69

6.6 Example 6

Treatment of Amyotrophic Lateral Sclerosis Using a Combined Stem CellPopulation

Amyotrophic Lateral Sclerosis (ALS), also called Lou Gehrig's disease,is a fatal neurodegenerative disease affecting motor neurons of thecortex, brain stem and spinal cord. ALS affects as many as 20,000Americans with 5,000 new cases occurring in the US each year. Themajority of ALS cases are sporadic (S-ALS) while ˜5-10% are hereditary(familial—F-ALS). ALS occurs when specific nerve cells in the brain andspinal cord that control voluntary movement gradually degenerate. Thecardinal feature of ALS is the loss of spinal motor neurons which causesthe muscles under their control to weaken and waste away leading toparalysis. ALS manifests itself in different ways, depending on whichmuscles weaken first. ALS strikes in mid-life with men beingone-and-a-half times more likely to have the disease as women. ALS isusually fatal within five years after diagnosis.

ALS has both familial and sporadic forms, and the familial forms havenow been linked to several distinct genetic loci. Only about 5-10% ofALS cases are familial. Of these, 15-20% are due to mutations in thegene encoding Cu/Zn superoxide dismutase 1 (SOD1). These appear to be“gain-of-function” mutations that confer toxic properties on the enzyme.The discovery of SOD mutations as a cause for ALS has paved the way forsome progress in the understanding of the disease; animal models for thedisease are now available and hypotheses are being developed and testedconcerning the molecular events leading to cell death.

Presented below is an example method of treating an individual havingALS with A combined stem cell population. The method involvesintravenous infusion through a peripheral, temporary angiocatheter.

An individual having ALS is first assessed by the performance ofstandard laboratory analyses. Such analyses may include a metabolicprofile; CBC with differential; lipid profile; fibrinogen level; ABO rHtyping of the blood; liver function tests; and determination ofBUN/creatine levels. Individuals are instructed the day prior to thetransplant to take the following medications: diphenhydramine(BENADRYL™), 25 mg t.i.d, and prednisone, 10 mg.

A combined stem cell population is produced from a unit of placentalperfusate and a matched unit of cord blood (that is, the perfusate istaken from the same placenta from which the cord blood is obtained).Total nucleated cell populations from the perfusate and the cord bloodare isolated, and samples of each are tested in vitro in a plurality ofratios to determine the ratio that produces the highest number ofcolony-forming units. The two populations are combined in approximatelythat ratio to create a combined stem cell population. This stem cellpopulation is maintained for approximately two days prior totransplantation at a temperature of about 5° C.

The individual is transplanted at an outpatient clinical center that hasall facilities necessary for intravenous infusion, physiologicalmonitoring and physical observation. Approximately one hour prior totransplantation, the individual receives diphenhydramine (BENADRYL™), 25mg×1 P.O., and prednisone, 10 mg×1 P.O. This is precautionary, and ismeant to reduce the likelihood of an acute allergic reaction. At thetime of transfusion, an 18 G indwelling peripheral venous line is placedinto one of the individual's extremities, and is maintained open byinfusion of D5 ½ normal saline+20 mEq KCl at a TKO rate. The individualis examined prior to transplantation, specifically to note heart rate,respiratory rate, temperature. Other monitoring may be performed, suchas an electrocardiogram and blood pressure measurement.

The combined stem cell population is then infused at a rate ofapproximately 1-2×10⁹ total nucleated cells per hour in a totaldelivered fluid volume of 60 ml. Based upon data from pre-clinicalstudies in mice, a total of 2.0-2.5×10⁸ cells per kilogram of bodyweight should be administered. For example, a 70 kilogram individualwould receive approximately 14-18×10⁹ total nucleated cells. Theindividual should be monitored for signs of allergic response orhypersensitivity, which are signals for immediate cessation of infusion.

Post-infusion, the individual should be monitored in a recumbentposition for at least 60 minutes, whereupon he or she may resume normalactivities.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

What is claimed is:
 1. A method of identifying a ratio of placental stemcells to stem cells from a second source, comprising combining andculturing a number of placental stem cells and a number of stem cellsfrom a second source in a plurality of ratios for a time and underconditions sufficient to allow colony-forming units to form, andidentifying the ratio in said plurality of ratios that produces agreater number of colony-forming units than a number of placental stemcells or stem cells from the second source, equivalent to the totalnumber of placental stem cells and stem cells from the second source,alone, and produces the highest number of colony-forming units in saidplurality of ratios.
 2. The method of claim 1, wherein said placentalstem cells are derived from a single placenta.
 3. The method of claim 1,wherein said placental stem cells are derived from a plurality ofplacentas.
 4. The method of claim 1, wherein said placental stem cellsare stem cells from placental perfusate.
 5. The method of claim 1,wherein said stem cells from the second source are cord blood-derivedstem cells.
 6. The method of claim 5, wherein said cord blood-derivedstem cells are hematopoietic stem cells.
 7. The method of claim 1,wherein said placental stem cells and the stem cells from the secondsource are combined in a suspension.
 8. The method of claim 7additionally comprising adding to said suspension a bioactive molecule.9. The method of claim 8, wherein said bioactive molecule is a cytokineor a growth factor.
 10. The method of claim 1, wherein said placentalstem cells are obtained from a single individual.
 11. The method ofclaim 1, wherein said placental stem cells are obtained from a pluralityof individuals.
 12. The method of claim 1, wherein said placental stemcells are CD34⁻ and not trophoblasts.
 13. The method of claim 12,wherein said placental stem cells are CD34⁻, CD10⁺, CD105⁺ (SH2⁺) andCD200⁺.
 14. The method of claim 13, wherein said placental stem cellsare additionally CD45⁻ and CD90⁺.
 15. The method of claim 1, whereinsaid stem cells from the second source are hematopoietic stem cells. 16.The method of claim 15, wherein said hematopoietic stem cells from thesecond source are CD34⁺.
 17. The method of claim 16, wherein said CD34⁺cells are isolated from umbilical cord blood (UCB).
 18. The method ofclaim 16, wherein said CD34⁺ cells are contained within UCB.